J. Am. Chem. SOC. 1995,117, 66-79
66
Ligand Recognition by E-Selectin: Synthesis, Inhibitory Activity, and Conformational Analysis of Bivalent Sialyl Lewis x Analogs Shawn A. DeFrees,? Winfried Kosch,? William Way,? James C. Paulson,*J3$ Subramanian Sabesan? Randall L. Halcomb,l Dee-Hua Huang,l Yoshitaka Ichikawa,l and Chi-Huey Wong*d Contribution from Cytel Corporation, 3525 John Hopkins Court, San Diego, Califomia 92121, Central Research and Development, DuPont Company, Wilmington, Delaware, and Department of Chemistry, The Scripps Research Institute, 10666 North Torrey Pines Road, La Jolla, Califomia 92037 Received May 26, 1994@
Abstract: Several sialyl Lewis x dimers anchored onto a galactose template or attached to 1A-butanediol or 1,5pentanediol have been prepared chemoenzymatically and evaluated as inhibitors of E-selectin-mediated cell adhesion. Two monosaccharide units were simultaneously incorporated (Le., Gal, NeuAc, Fuc) by a glycosyltransferase into a chemically synthesized core structure containing GlcNAc and Gal. Each of the galactose-anchored dimers had higher activity than the sialyl Lewis x pentasaccharide la, with the general trend being 3,6-linked > 2,3 2 4,6 2 2,6 > monomer. The dimers linked to butanediol or pentanediol showed the same level of activity as the pentasaccharide monomer. Conformational analysis of these dimers with NMR indicated that each sialyl Lewis x domain of the dimers retains the same conformation as the monomer. The differences in activity of the dimers most likely derive from differences in the relative orientation and distance between the monomer domains, suggesting the importance of the linker used in the preparation of dimers.
Carbohydrate-mediated cell adhesion is an important event initiated by tissue injury or infection and is involved in cancer metastasis.' One such recently discovered adhesion process is the interaction between the glycoprotein E-selectin (formerly called endothelial leukocyte adhesion molecule- 1 or ELAMlle) on the surface of activated endothelial cells and an oligosaccharide structure displayed on the surface of neutrophils (Figure 1). The ligand recognized by E-selectin has been identified as the tetrasaccharide sialyl Lewis x (SLeX, la).* Recent studies of the inhibition of E-selectin indicate that, in addition to SLex,2a several related structures including the glucose analog lb,3asialyl Lewis A 2,3bLex-3'-O-sulfate 3,3c and sialyl LeX glycal 43d(Figure 2a) have similar inhibition + Cytel Corp.
DuPont Co. The Scripps Research Institute. Abstract published in Advance ACS Abstracts, December 1, 1994. (1) (a) Paulson, J. C. In The Receptors; Conn, M., Ed.; Academic Press: New York, 1985;Vol. 2,pp 131-219. (b) Paulson, J. C. In Adhesion: its role in inflammatory disease; Harlan, J., Liu, D., Eds.; W. H. Freeman: New York, 1992;Chapter 2, p 19. (c) Springer, T. A.; Lasky, L. A. Nature 1991,349, 196. Lasky, L. A. Science 1992,258,964. (d) Rice, G. E.; Bevilacqua, M. P. Science 1989,246,1303. (e) Bevilacqua, M. P.; Pober, J. S.; Hendrick, D. L.; Cotran, R. S.; Gimbrone, M. A. Proc. Natl. Acad. Sci. U S A . 1987,84, 9238. (f) For cDNA of E-selectin: Bevilacqua, M. P.; Stengeling, S.; Gimbrone, M. A., Jr.; Seed, B. Science 1989,243,1160. (2)(a) Phillips, M. L.; Nudelman,E.; Gaeta, F. C. A.; Perez, M.; Singhal, A. K.; Hakomori, S.; Paulson, J. C. Science 1990,250, 1130. (b) Waltz, G.:Amfto. A.: Kalanus. W.: Bevilacaua. M.: Seed. B. Science 1990. 250. 1132. (c) Lowe, J. B.; Stoolman, L. M:; Nair, R. P.; Larson, R. D.; Berhend, T. L.; Marks, R. M. Cell 1990,63,475. (3) (a) Tyrrell, D.; James, P.; Rao, N.; Foxall, C.: Abbas, S.: Dasmuta. F.; Nashed,-M.; Hasegawa, A.; Kiso, M.; Asa, D.; Kidd, J.; Brandley; B. K. Proc. Natl. Acad. Sci. U.SA. 1991,88,10372.(b) Berg, E. L.; Robinson, M. K.; Mansson, 0.; Butcher, E. C.; Magnani, J. L. J . Biol. Chem. 1991, 266, 14869. (c) Yuen, C.-T.; Lawson, A. M.; Chai, W.; Larkin, M.; Stoll, M. S.; Stuart,A. C.; Sullivan, F. X.; Ahem, T. J.; Feizi, T. Biochemistry 1992,31,9126.(d) DeFrees, S.A.; Gaeta, F. C. A.; Lin, Y.-C.; Ichikawa, Y.; Wong, C.-H. J . Am. Chem. SOC. 1993,115,7549. 0
@
activities for E-selectin binding, with the ICs0 values in the range of 1-2 mM3dbased on an ELISA assay. Conformational analysis of 1-4 indicates that these oligosaccharides possess a common topostructure in the space composed of NeuSAc, Gal, and F U C , suggesting ~~.~ that the E-selectin binding domain of sialyl Lexmay reside on the surface illustrated by 5. The bivalent sialyl Lex(6)is, however, about 5-fold better than SLex as an inhibitor of E-selectin, suggesting a possible multivalent ligand-receptor i n t e r a ~ t i o n .This ~ ~ is further supported by the observation in a preliminary study that the conformation^^^ of the two SLeXunits are essentially the same as monomeric SLex. Although the affinity of SLexfor E-selectin is relatively weak in vitro, the IC50 in vivo for protecting against lung injury in rats is about 1 pM? suggesting that the inhibition analysis in vitro does not reflect the activity in vivo. This high in vivo activity and the available large-scale chemo-enzymatic synthesis4 of SLeX,however, has led to the development of SLex as a potential new antiinflammatory agent.5 Since the bivalent SLex derivative is more active than the monomer investigations of the activities of other multivalent analogs were undertaken with the hope that more effective antagonists could be developed. As part of our interest in further understanding the nature of E-selectin ligand recognition, we report here the syntheses of several bivalent SLeX analogs with different spacer groups and analyses of their inhibition activities. Efforts to correlate
(4)Ichikawa, Y.; Lin, Y.-C.; Dumas,D. P.; Shen, G.-J.; Garcia-Junceda, E.; Williams, M. A,; Bayer, R.; Ketcham, C.; Walker, L. E.; Paulson, J. C.; Wong, C.-H. J . Am. Chem. Soc. 1992,114,9283.Both galactosylation and sialylation have been carried out on 0.5-1 kg scales using galactosyltransferase and sialyltransferase coupled with regeneration of the corresponding nucleoside phosphates. The fucosylation step is currently performed chemically in large-scale processes. Work is in progress to prepare recombinant .GDP-fucose pyrophosphorylase for the large-scale regeneration of GDP-fucose. ( 5 ) Mulligan, M. S.; Paulson, J. C.; DeFrees, S.; Zheng, Z.-L.; Lowe, J. B.; Ward, P. A. Nature 1993,364, 149.
0002-7863/95/1517-0066$09.00/0 0 1995 American Chemical Society
J. Am. Cltem. Soc.. Vol. 117. No. 1. 1995 61
Ligand Recognition b j E-Selecrin
E-Selectin M e d i a t e d Cell A d h e s i o n
Inflammatory Reaction Involves A. /n&v
Occurs
E. Cyrokines Refease4 E-Sefeclins Produced
C. Adhesion Occurs
D. White Bfood Cell Ro//ing
E. furiher Adhesion Mea7ated
F. White 8fOod C e h Reach /"/UT
w.0 -
by Intqrins, Extravasation RGD hpnd /CAM-t
';./"/qd"
*
Sfte
%/-%
F i p m 1. Inflammatory reaction involves E-selectin-mediated adhesion. When tissue injury OCCUR (AI. cytokiner are released to signal E-selectin synthesis (B). Adhesion then occurs by interaction of E-selectin and SLe' on leukocytes. As leukocytes roll along the surface of endothelial cells. funher adhesion occurs by interactions between integrins on leukocytes and the Arg-Gly-Asp (RDC)-containingligand within intercellular adhesion molecule (ICAM-I) to enable leukocytes to reach the site of injury. If too many leukocytes are recruited to the site of injury. normal cells can also be damaged. activity with 3-dimensional conformation, as determined by NMR. are also described.
Resulh and D i s c u s i o n A. Synthesis of Dimeric Sialyl Lewis X Derivatives. The dimeric sialyl Lewis X derivatives were synthesized using a combined chemical and enzymatic r o ~ t e . ) ~Small . ~ acceptor primers were chemically synthesized, and the peripheral carbohydrate residues were added enzymatically. The synthesis of the primer for the dimer 6. which contains a somewhat rigid galactose unit as a template. is shown in Scheme 1. The was selectively benzoylated protected galactose derivative 77.R at 0-Z1 in a three-step procedure to give 8.R which was subsequently glycosylated with a phthalimido-protected glucosamine to give the disaccharide 9. The benzylidene was hydrolyzed to give the diol 10. which was regioselectively glycosylated at 0-6 to give the trisaccharide ll.l''.ll Hydrazinolysis of 11 to remove the phthalimide and ester protecting groups followed by acetylation gave compound 12. Finally, saponification of the 0-acetates provided 13.R the primer for the following enzymatic glycosylations. (6)For recent reviews on chcmc-enzymatic synthesis of complex cahhydraten: la) Tonne. E. 1.: Simon. E. S.:Bednmki. M.D.: Whiterider. G. M. Terrohcdron 1989. 45. 5365. (b) Dmeckhammer. D. G.: Hennen. W. J.: Pedenon. R. L.: Bahbar. C . F.. 111: Gautheron. C. M.: Kmch. T.: Wang. C.-H. Sjnrherir 1991,499. (c) Hindrgaul. 0.Sem. C ~ l l B i o l1991. . 2.319. (d) Look. G. C.; Ichikawa. Y.: Wong. C.-H.Ana1. Bimhem. 1992. 202. 215. (e) 110. Y.: Gaudina. J. J.: Paulron. J. C. PWP Appl. Chm. 1993. 65. 7 5 3 . (0 Wong. C.-H:. Hatcomb. R. L.: Ichikawa Y:. K a j k " . T. A ~ R ~ Chon.. w. btr. Ed. h ~ l . in, press. (7) Wallenfelr. von K.: Lehmann. J. LiehipsAnn. Chon. 1960.635. 166. ( 8 ) Lemieux. R. U.: Abhas. S. Z.: Chung. B. Y. Can. 1. Chem. 1981. 60. 68. (9)The position of the ?-benzoate in 8 wa.. verified by adding trichlomacelyl isocyanate to the NMR sample (CDCh) to form Sa. The Spectrum contained a doublet of J doublet at 5.27 ppm ( J = 8.0 and 2.3 H7.l typical of the H-3 of galactme. (101 Lemieua. R. U.: Takcda. T.: Chung. B. Y. ACS Symp. Ser. 1976. 39.90. (1 I)Thhe position of substitution was venfied by reacting 11 with tnchlomacctyl isocyanate in the NMR tube (CDCId to pmduce Ila. A dounfield shift of a new peak to 5.39 ppm with two small coupling consmts ( J = 2. I Hzi was observed indicating that the 4-fmition pmton of the ethyl galactoside was unsubnituted.
Scheme 2 illustrates the synthesis of the dimeric SLe" 6'd from 13. Treatment of 13 with ~-l.4-galactosyltransferaseand 2 equiv of UDP-Gal gave the pentasaccharide 14. In a similar manner. two sialic acid residues were added with N-type a-2.3sialyltransferase (EC 2.4.99.6) to give 15 (RO% isolated yield). Alkaline phosphatase was added to hydrolyze the product phosphoester C M P and thus drive the reaction to completion.12 Monitoring the reaction by TLC suggested that the incorporation of the first sialic acid was rapid. but that the incorporation of the second was relatively slow. Finally. two fucose residues were added with a-1.3-fucosyltransferase V" to give 6 as the ammonium salt (84% isolated yield). The dimers 16-19 (Figure 2b) were prepared by doubly sialylating and fucosylating previously reponed dimeric N-acetyllactosamine derivatives (see Experimental Section)." It is noted that fucosylation of 36 only gives a mixture of mono- and difucosylated products which are not separable. Compound 19 was therefore not evaluated for its activity. The SLex dimers having flexible butanediol and pentanediol templates were synthesized in an analogous manner. The chemical synthesis of the requisite primers is shown in Scheme 3. Glycosylation of either 1.4-butanediol or 1.5-pentanediol with 2 equiv of acetobromogalactose gave 20a and 20h. respectively. Deacetylation afforded 2la and 21h. each of which was regioselectively acetylated at the 2- and &positions of galactose. vin the intermediate isopropylidene.I4 to give 2Za and 22b. respectively. Glycosylation of 22a with 2 equiv of 3.4.6-triO-acetyl-2-deoxy-2-phthalimid~~glucopyosyl bromide gave the bis(disaccharide) 23. Similarly. glycosylation of 22h with a protected lactosamine donor provided 24. Hydrazinolysis and acetylation of 23 gave 25. which was then deacetylated to give 26 (Scheme 4). Sequential enzymatic (12) (a) Saberan. S.: Paulson. J. C. 1. Am. Chcm. Sor. 1%. 108.2068. (b) Unverwgt. C.: Kunr. H.: Paulron. J. C. 1. Am. Chcm. Sot. 1990. 112. 930% (13) Weston. B. W.: Nair. R. P.: h e n . R. D.: Lowe. 1. B. J. B i d . C h m . 1992. 267.4152. (I4)Sabe~an.S.:Duus.J.O.;Ncin.S.:Domaille.P.:Kelm.S.:Paulran. J. C.: Bock. K. J. Am. Chew. Sor. 1992. 114. 8363. (15)(a) Paulren. H.: Paal. M. Cwhohydr. R ~ Y .157%. 137. 39. (b) Paulsen. H.: Pad. M.: Hadamcryk. D.: Steiger. K:M. Carhohydr. Res. 19R4. 131. CI.
DeFrees et al.
68 J. Am. Chem. SOC., Vol. 117,No. 1, 1995 HO I OH HO * o
e
O
R
.O OH HO
OH
3 Lex3-O-sulfate
la: R' = NHAC (sialg Lex) l b : R' = OH
no
I OH
no
no
I OH
OH
no
4
S
Sialyl LeXgiycai
Active binding site
HO I OH
HO I OH
HO I OH
no
I OH
OH
Figure 2. Ligands for E-selectin (1-6) and (b) Sialyl Lewis x dimers (16-19). transfer of galactose, NeuAc, and fucose residues gave 29 via B. Biological Activity of SLeX Dimers. A soluble form of E-selectin (sol-E-selectin) was prepared for inhibition assays the intermediates 27 and 28. Similarly, compound 24 was of the dimeric SLex derivatives. A 1.67 kbp DNA fragment converted to the dimer 33 via the bis(tetrasaccharide) 32 (not encoding a truncated structural gene for E-selectin was isolated shown) as outlined in Scheme 5 .
Ligand Recognition by E-Selectin
J. Am. Chem. Soc., Vol. 117, No. I, 1995 69
Scheme 1" 4
0
4
HO
8_
OH
H O k OBz o E t
b_
0
G - O k ONPht E t OBz
7
8
9
Q
'NRt
'OBz
NPht
10
A%
OBr
11
&
0
AcHN ACO
AE-CI&OEt
NHk
OAc
&
OEl
NHAc
12
OH
13
Key: (a) (i) (ClCHzCO)zO, pyridine, CHzC12, -65 "C; (ii) PhCOC1, -65 to 25 "C; (iii) NH3 i n MeOH, MeOH, -30 "C (48% overall); (b) 2-deoxy-2-phthalimido-2,4,6-~-O-acetyl-~-~-glycopyranosyl bromide, AgOTf, collidine, CH2C12, -20 OC (86%); (c) AcOH, Hz0, 80 OC (80%); (d) 2-deoxy-2-phthalimido-2,4,6-tri-O-acetyl-~-~-galactop~anosyl bromide, AgOTf, collidine, CHzClz, -20 "C (86%); (e) (i) H2NNH2-H20, EtOH, reflux; (ii) AczO, pyridine, DMAP (76%); (0 NaOMe, MeOH (70%).
Scheme 2"
b
13
HO
HO
OEt
C
6
15
no
Key: (a) UDP-glucose, uridine 5'-diiphosphogalactose 4'-epimerase, B- 1,Cgalactosyl transferase, sodium cacodylate (0.2 M, pH 7 3 , MnClz (85%); (b) CMP-N-acetylneuraminicacid, a-2,3-sialyltransferase, sodium cacodylate (0.2 M, pH 6.5), MnC4 (82%); (c) GDP-fucose, fucosyltransferase V, sodium cacodylate (0.2 M, pH 6.5), MnClz (84%). by PCR amplification of cDNA derived from messenger RNA that was isolated from IL-1 activated human endothelial cells. The cDNA was subcloned into the vector pBluescript II and was transfected into 293 cells. The clones were screened for the production of sol-E-selectin, and the clone 293#3 was selected as the stable cell line that produced the greatest amount of sol-E-selectin per cell. Sol-E-selectin was produced on a large scale from this line using a Nunc cell factory. Recom-
binant sol-E-selectin was isolated from the media using immunoaffinity chromatography. The SLeX dimers were assayed for ability to block the adhesion of HL-60 cells to immobilized sol-E-selectin. Immobilized E-selectin was incubated f i s t with inhibitor and then with HL-60cells. The bound cells were lysed, and myeloperoxidase released from the bound cells was detected with o-phenylenediamine and hydrogen peroxide. The percentage
DeFrees et al.
70 J. Am. Chem. SOC.,Vol. 117, No. I, 1995
Scheme 3a ~ c o OAc
ACO
a
OAc Br
Ad)
AhC -O 0OAC G ,O T ' "
+
b
Aco
-
no OH
, O "H G 0 I ; " "
C
HO
HO-(CH2),-OH
OH
OAc
218 (n = 4)
2Oa (n = 4) 20b (n = 5)
2lb (n = 5)
HO
OAC A%&&o&&o,NPht
NPhl
t
(n = 4)
OAc
m&rGoy
A&&,ACO ,
Aco NPhl
d -
(n = 5) 22. (n = 4)
OAc
23
A&
-
OAc
NPht
22b (n = 5) OAc
OAC
NPhl
24
Key: (a) Ag2C03, CH2C12, -20 "C (68-73%); (b) NaOMe, MeOH (92%); (c) (i) 2,2-dimethoxypropane, acetone, DMF, reflux; (ii) AczO, pyridine; (iii) AcOH, H20, reflux (28-38% overall); (d) AgOTf, CH2C12, -20 "C (23: 52%, 24: 40%).
Scheme 4"
'OAC
NHAc
1
.
OAC
NHAc
'OH
NHAc
26
25
HO OH
29
Key: (a) (i) H~NNHz-H~O,EtOH, reflux; (ii) AczO, pyridine (57%); (b) NaOMe, MeOH (96%); (c) (i) 2 equiv of UDP-glucose, uridine 5'-diiphosphogalactose 4'-epimerase, /%1,4-galactosyltransferase (27: 91%); (ii) 2 equiv of CMP-N-acetylneuraminicacid, a-2,3-sialyltransferase (28: 93%); (iii) 2 equiv of GDP-fucose, a-1,3-fucosyltransferase(29: 93%). a
inhibition was determined by comparing the absorbance of the resulting solution at 492 nm to that in wells containing no inhibitor.
Each dimer tested contained an SLe" domain linked to each of two positions of a galactose template. A pentasaccharide consisting of an SLeX linked to the 3 position of ethyl
J. Am. Chem. SOC.,Vol. 117, No. 1, 1995 I1
Ligand Recognition by E-Selectin Scheme Sa
As has been demonstrated in other ~ y s t e m s , ~multivalent ~J~ display of carbohydrate ligands often results in higher affinities for target protein receptors. The same principle appears to be true for the SL,eX-E-selectin interaction. Dimeric SLex derivatives, as illustrated here, can have affinities for E-selectin which are up to 5-fold greater than that of the monomer. There are also subtle differences in the inhibitory properties within the series of dimers, so factors other than simple multivalency must also be considered. Perhaps some of the dimers (e.g., 29 and 33) still exhibit monovalent activity. In the case of hemagglutinin inhibition, dimeric sialic acids exhibited 0- 10-fold increased inhibitory activity, depending on the nature of the linker used in the design.14 It is, however, noted that the assay used in hemagglutinin inhibition analysis is quite different from the E-selectin assay, which is not read out as a plus or minus for each data point. Each data point in the E-selectin assay is a direct measure of cells bound using a quantitative enzyme assay. The values were then plotted to give the titration curve and IC50 values. Five assays were performed for each inhibitor, and the results were highly reproducible. The standard deviation did not exceed f 6 . 3 % for any data point. The IC50 value reported here is derived from all the data, not by a titration end point as in hemagglutinin.
30
C. Conformational Analysis of S L e Dimers by NMR.
I OH
no
33
'Key: (a) (i) H M z - H z O , EtOH, reflux; (ii) Ac-20, pyridine (86%); (b) NaOMe, MeOH (96%); (c) (i) 2 equiv of CMP-Nacetylneuraminic acid, a-2,3-sialyltransferase (32: 97%); (ii) 2 equiv of GDP-fucose, a-1,3-fucosyltransferase(33: 89%). 100
80
60
40
20
0 1
1
10
LIGAND CONCENTRATION, mM
Figure 3. Inhibition of HL-60 adhesion to recombinant soluble E-selectin-coated plates: (0)pentasaccharide, NeuAca2,3-G@1,4(Fucal,3-)GlcNAc~1,4-G@OEt; (A)3,6-dimer 6; (A)4,6-dimer 16; (0)2,6-dimer 17; (0)2,3-dimer 18. Compounds 29 and 33 exhibited similar activities as the pentasaccharide. /?-galactoside was used as a monomer standard. As is shown in Figure 3, each of the five dimers was more potent than the monomer in blocking adhesion. The 3,6-linked SLe" dimer 6 was the most potent, with the general trend being 3,6-linked > 2,3 I 4,6 > 2,6 > monomer. The dimers attached to 1,4-butanediol (29) or 1,5-pentanediol (33) exhibited the same level of activity as the monomer. The concentrations of the dimers required to block adhesion of 50% of the cells ranged from 0.2 mM (for 6) to 0.45 mM. As references, the value for the pentasaccharide monomer was 0.8 mM and that for the tetrasaccharide monomer was 1.2 m M . 3 d 9 4
We have previously reported preliminary evidence that each SLex domain of 6 exists in the same conformation as in la. The same phenomenon was predicted for each of the other dimers. Therefore, it was tempting to speculate that the differences in the inhibition properties of each dimer are due to differences in the relative orientation of the SLe" domains in each respective dimer rather than changes in these domains. Validation of this hypothesis, however, requires a detailed conformational analysis of each compound. As a first step toward this objective, we have carried out NMR studies of selected dimers. We report the complete assignment of the proton and carbon resonances of 6 and some 2D NMR experiments with 16 and 17. Although the 1D 'H NMR spectrum of the dimer 6 was quite complex, certain key proton resonances were sufficiently resolved so as to allow the use of various 2D techniques to make a complete proton and carbon assignment. Protons were assigned in a manner similar to that reported for the SLeX monomer: making use of PE-COSY, ROESY, TOCSY, HMl3C, and HMQC techniques. The anomeric proton of the template Gal was identified by the presence of an ROESY to one of the methylene protons of the aglyconic ethyl group. This permitted identification and assignment of all of the protons of this Gal spin system. Each of the GlcNAc anomeric protons was identified by the presence of ROE'S to the template Gal residue, particularly an ROE between H-1 of the 3-0-GlcNAc and H-3 of the template Gal, and between H-1 of the 6-0-GlcNAc and H-6 of Gal. After identification of these key protons, all of the resonances of each GlcNAc spin system were assigned using HMBC, HMQC, and COSY (Figures 4 and 5). The protons of the Fuc, Gal, and NeuAc residues of the SLeXdomains were assigned in a similar manner, employing techniques previously used by our group (16) (a) Spaltensin,A,; Whitesides,G. M. J. Am. Chem. SOC.1991,113, 686. (b) Glick, G. D.; Toogood, P. L.; Wiley, D. C.; Skehel,J. J.; Knowles, J. R. J. Biol. Chem. 1991, 266, 23660. (c) Conolly, D. T.; Townsend, R. R.; Kawaguchi, K.; Bell, W. R.; Lee, Y. C. J . Biol. Chem. 1982,257,939. (d) Weis, W. J.; Drickamer, K.; Hendtickson, W. A. fluture 1992, 360, 127. (e) Kingery-Wood, J. E.; Williams, K. W.; Sigal, G. B.; Whitesides, G. M. J . Am. Chem. SOC. 1992, 114, 7303. (f) Spevak,W.; Nagy, J. 0.; Charych, D. H.; Schaefer, M. E.; Gilbert, J. H.; Bednarski, M. D. J. Am. Chem. SOC. 1993, 115, 1147. (g) Roy, R.; Pon, R. A.; Tropper, F. D.; Andersson, F. 0. J. Chem. Soc., Chem. Commun. 1993, 264.
DeFrees et al.
12 . I Am. . Chem. SOC., Vol. 117, No. 1, 1995
1
1.o
2.0
3.0
5.0
4.0
4.4
ERI
D1 ( P P W
I
1
4.0
3.6
(ppm)
Figure 5. PECOSY spectrum of 6. 3.6
3.8
4.0
D2 4.2
18 Vi
Gal H-1
Gal-OEI
- H1-H2
H-1
i
GlcNac2H-l-HZ
GlcNAc
4.6
4.4
4.2
-1.8
H.2
4.0
0 I1
-
Gall +Gal2 Hl-HZ
1
3.8
(PPm)
Gal H-2
4*6
3.6
D1 (PPm)
Figure 4. (Top) 2D phase-sensitive double quantum filter COSY (COSYDFPT) of 6. (Bottom) expansion of 2D COSYDFPT of 6. to assign the monomef' (see Table 1 for complete assignment). No attempt was made to distinguish between the corresponding resonances of each of the two Fuc, Gal, and NeuAc residues due to the coincidence of most of the signals. One conclusion that can be drawn upon examination of the ROESY spectrum of 6 (Figure 6) is that each SLex domain exists in basically the same conformation as monomeric SLex. The presence of cross peaks between Fuc H-5 and Gal H-2, between Fuc H-6 and Gal H-2, and between NeuAc H-2, and Gal H-3 are consistent with those observed for the monomer. Additionally, several ROESY cross-peaks from each GlcNAc to the template Gal were apparent. A detailed analysis of these cross peaks should permit the determination of the relative orientation and distance between each respective SLeXdomain and is the subject of current study. We have carried out experiments with dimers 16 and 17. The ROESY spectra of 16 and 17 (data not shown) suggest that the SLeX regions of these compounds also exist in the same conformation as la, thus supporting our hypothesis. Summary. In conclusion, several SLeX dimers were synthesized and evaluated as inhibitors of E-selectin-mediated cell
adhesion. Each of the dimers had greater activity than l a according to the assay used, with the general trend being 3,6linked > 2,3 1 4,6 > 2,6 > monomer. No increase of activity was observed for the dimers linked by butane- or pentanediol. A general conclusion that can be drawn from these results is that multivalent display of the SLe" ligand leads to increased activity in certain cases. There are, however, slight differences in the activity of each dimer. On the basis of NMR analysis, each SLe" domain of 6 retains the same conformation as the monomer. The results of inhibitory and conformationalanalysis indicate that 6 interacts with two adjacent E-selectin molecules. The same situation is predicted for each of the other dimers based on our inhibitory analysis and preliminary NMR results. Therefore, the differences in activity of the dimers most likely derives from differences in the relative orientation and distance between the SLeXdomains. How changes in this orientation and spacing are manifested in differences in the mode of inhibition of cell adhesion remains to be determined, and are the subject of ongoing research.
Experimental Section Synthesis. General. All reactions were monitored by thin layer chromatography carried out on 0.25 mm Whatman silica gel plates (60F254) using UV light and anisaldehyde reagent as developing agent (Gordan, A. J.; Ford, R. A. The Chemists Companion; WileyInterscience: New York, 1972; p 377). E. Merck silica gel (60, particle size 0.040-0.063 mm) was used for flash column chromatography. All reactions were carried out under an argon atmosphere and with anhydrous solvents from Aldrich unless otherwise noted. Yields refer to chromatographicallyand spectroscopically ('HNMR) homogeneous materials unless otherwise stated. The UDP-glucose, CMP-sialic acid, uridine 5'diphosphogalactose 4-epimerase, and ,81,4-galactosyltransferase were purchased from Sigma. The GDP-#I-fucose was purchased from Oxford Glycosystems. The N-type a(2,3)-sialyl transferase and fucosyl transferase V enzymes were prepared at Cytel Corp. Ethyl 4,6-0-Benzylidene-2-O-benzoyl-~-~-galactopyr~oside (8) and Ethyl 4,6-0-Benzylidene-3-O-((trichloroacetyl)-N-carbamoyl)Z-O-benzoyl-/3-D-galactopyranoside (8a). Ethyl 4,6-U-benzylidene,8-D-gdactopyranoside (7)' (2.9 g, 9.8 "01) was dissolved in CHzClz (29 mL) and pyridine (5.4 mL) and cooled to -65 OC under an argon was then atmosphere. Chloroacetic anhydride (0.82 mL, 10.3 "01) added and the reaction mixture stirred at -65 "C for 2 h. The first stage of the reaction was complete at this time, and thus benzoyl chloride (1.36 mL, 11.78 "01) was added at -65 "C. The reaction
Ligand Recognition by E-Selectin
J. Am. Chem. SOC.,Vol. 117, No. I, 1995 73
Table 1. Complete Chemical Shift Assignment of 6 NeuAc carbon no. I
H
2
Gal C 1.73 1.73
H 4.48 4.48
100.28 100.28
3.47 3.47
Fuc C 102.0 102.1
GlcNAc
branch Gal
H 5.06 5.05
C 99.2 99.2
H 4.50 4.65
C 103.1 103.1
H
C
4.30
69.95 69.95
3.62 3.62
67.3 67.3
3.87 3.89
55.3 55.3
3.45
70.06
103.0
3
1.74, 2.70 1.74, 2.70
40.3 40.3
4.03 76.3
76.3 3.83
3.83 68.8
68.8 3.83
3.83 74.5
74.5
3.63
82.86
4
3.63 3.63
69.0 69.0
3.88 3.88
66.9 66.9
3.71 3.72
71.5 71.5
3.93 3.93
73.71 73.71
4.06
69.1
5
3.80 3.80
52.2 52.2
3.51 3.51
75.58 75.58
4.77 4.77
67.3 67.3
3.54 3.54
68.7 68.7
3.71
70.2
6
3.65 3.65
72.6 72.6
3.63 3.63
62.08 62.08
1.10 1.10
15.8 15.8
3.82,3.85 3.82, 3.85
60.2 60.2
3.70, 3.97
70.13
7
3.59 3.59
67.7 67.71
8
3.90 3.90
71.4 71.4
9
3.59, 3.82 3.59, 3.82
63.7 63.7
CH3
2.03 2.03
21.7 21.7
2.01 2.01
21.9 21.9
OCHzCHs 3.65 3.88
66.59
1.17
14.8
c=o
175
4: 4
4:O 3:6 D I M E R (ppm)
Figure 6. ROESY spectrum of 6 (mixing time 350 ms). mixture was allowed to warm to room temperature. The mixture was stirred overnight and then diluted with CHzClz (100 mL) and washed with 1 M citric acid (100 mL), water (50 mL), saturated NaHC03 (50 mL), water (50 mL), and saturated NaCl(50 mL). The organic layer was dried (NazS04), filtered, and concentrated to afford crude 8. This material was then dissolved in methanol (50 mL) and cooled to -30 OC. A solution of 2 M NH3 in methanol (6.0 mL) was added, and the reaction mixture was stirred at -30 "C for 4 h. The resulting mixture was poured into CHZClZ (100 mL) and washed with water (100 mL), saturated NaHC03 (100 mL), water (100 mL), and saturated NaCl(50 mL). The organic layer was dried (Na~S04),filtered, concentrated, and chromatographed (silica, 80% ethyl acetatelhexane) to afford 1.7 g (48%) of 6 as a white solid: Rf = 0.3 (silica, ethyl acetatelhexane); 'H NMR (CDC13) 6 8.07 (d, J = 7.5 Hz, 2H, arom), 7.55 (m, 3H,
173.7 173.4
arom), 7.46 (d, J = 7.5 Hz, 2H, arom), 7.38 (m, 3H, arom), 5.59 (s, lH, benzylidene), 5.36 (dd, J = 10.07, 8.2 Hz, lH, H-2), 4.64 (d, J = 8.2 Hz, lH, H-l), 4.39 (dd, J = 12.5, 1.1 Hz, lH, H-6), 4.27 (d, J = 4.0 Hz, lH, H-4), 4.12 (dd, J = 1.5, 12.5 Hz, lH, H-6), 3.99-3.86 (multiple peaks, 2H, OCHzCH3, H-3), 3.61 (m, lH, OCHZCH~), 3.56 (bs, lH, H-5), 2.63 (d, J = 9.0 Hz, lH, OH), 1.21 (t, 3H, CH3). Trichloroacetyl isocyanate was then added to the NMR sample to f o m 8a. A new low-field signal was observed at 5.27 ppm (J = 3.1, 9.7 Hz) typical for the H-3 of galactose. This c o n f i i e d that the H-3 position of 8 was unsubstituted. Ethyl 4,6-0-Benzylidene-2-O-benzoyl-3-0-(3,4,6-tri-O-acetyl-2d e o x y - 2 - p h t h ~ d o ~ - ~ - ~ u c o p y r a n o s y l ) ~ - ~ g ~(9). ctop~~~de A suspension of molecular sieves (4 A, 1 g), collidine (0.167 mL, 1.26 mmol), silver triflate (0.3 g, 1.16 mmol), and 8 (0.71 g, 0.974 "01) in CHzClz (10 mL) was stirred under argon for 1 h. The reaction mixture was then cooled to -20 "C and a solution of 3,4,6-tri-O-acetyl2-deoxy-2-phthalimido-~-~-glucopyranosyl bromide3 (0.53 g, 1.07 mmole) in CHzClz (1 mL) was added. The reaction mixture was stirred at -20 "C for 30 min, allowed to warm to room temperature, and fdtered through a Celite pad. The filtrate was diluted with ethyl acetate (100 mL), washed with water (50 mL), 1 M citric acid (100 mL), water (50 mL), saturated NaHCO3 (100 mL), water (50 mL), and saturated NaCl (50 mL), dried (NazS04), and concentrated. The residue was purified by chromatography (silica, 40% ethyl acetate/toluene) to afford 0.967 g (86%) of 9 as a white solid Rf = 0.4 (silica, 70% ethyl acetate/ hexane); 'H N M R (CDC13) 6 7.65 (dd, J = 2.9, 7.5 Hz, 2H, arom), 7.54(dd,J=7.6, 1.8Hz,2H,arom),7.50(dd,J=7.0,7.0,2H,arom), 7.45-7.31 (m, 6H, arom), 7.28 (d, J = 9.7 Hz, lH, arom), 7.25 (d, J = 7.1 Hz, lH, arom), 5.65 (dd, J = 9.4, 10.8 Hz, lH, H-3 GlcN), 5.61 (d, J = 8.6 Hz, lH, H-1 GlcN), 5.57 (s, lH, benzylidene), 5.38 (dd, J 8.0, 10.1 Hz, lH, H-2 Gal), 5.16 (dd, J = 9.6, 9.6 Hz, lH, H-4 GlcN),4.52(d,J=8.OH~,lH,H-lGal),3.96(dd,J=3.6, lO.lHz, lH, H-6 GlcN), 3.87-3.77 (m, 2H), 3.48-3.43 (m, 2H, OCHzCH3 and H-5 Gal), 2.07 (s, 3H, OAC),2.01 (s, 3H, OAC), 1.76 (s, 3H, OAC), 0.951 (t, 3H, CHzCH3). Ethyl 2-O-Benzoyl-3-0-(3,4,6-tri-O-acetyl-2-deoxy-2-phthabido~-D-g~ucopyranosy~)-~-D-gdactopyranoside (10). The benzylidene derivative 9 (1.0 g, 1.22 mmoles) was dissolved in a 80% aqueous acetic acid (20 mL), and the reaction mixture was heated at 80 "C
74 J. Am. Chem. SOC.,Vol. 11 7, No. 1, 1995
DeFrees et al.
aqueous acetic acid (20 mL) and then was heated at 80 "C for 30 min. Ethyl 3,6-B~-O-(2-ace~do-2-d~~-~-~-glucopyranosyl)-~-~ The reaction mixture was the poured into a solution of saturated galactopyranoside (13). Sodium methoxide (1.0 mL, 25% solution NaHC03 (200 mL), and solid NaHC03 was added until the pH was in MeOH) was added to a solution of trisaccharide 12 (0.366 g, 0.38 neutral. The solution was extracted with ethyl acetate (100 mL), and "01) in MeOH (40 mL). The reaction mixture was stirred for 20 h the organic layer was washed with water (2 x 100 mL) and saturated during which time a precipitate formed. Water (-5 mL) was added NaCl (20 mL). The organic layer was dried (NazS04), concentrated, until all of the solid had dissolved, and then prewashed BioGel AG and chromatographed (silica, 85% ethyl acetateihexane) to afford 0.716 50W-X8 (hydrogen form) was added to the reaction mixture until the g (80%) of diol 10 as a white solid Rf = 0.23 (silica, 80% ethyl acetate/ pH was neutral. The reaction mixture was then filtered, and the filtrate hexane); 'H NMR (CDC13) 6 7.58 (d, J = 7.9 Hz, 2H, arom), 7.43was concentrated. The residue was dissolved in water (10 mL) and 7.25(bm,5H,arom),7.18(d,J=7.7Hz, lH,arom),7.15(d,J=7.7 filtered through a C-18 sep pack (Whatman), and the fitrate was Hz, lH, arom), 5.66 (dd, J = 10.5, 9.3 Hz, lH, H-3 GlcN), 5.59 (d, J concentrated to yield 0.167 g (70%) of 13 as a white solid: Rf= 0.47 = 8.5 Hz, lH, H-1 GlcN), 5.29 (dd, J = 9.3, 9.3 Hz, lH, H-2 Gal), (silica, 30% 1 M NH40Acl2-propanol); 'H NMR (DzO) 6 4.64 (d, J = 5.11 (dd, J = 9.6, 9.6 Hz, lH, H-4 GlcN), 4.46 (d, J = 8.0 Hz, lH, 8.4 Hz, lH, H-1 GlcNAc), 4.48 (d, J = 8.4 Hz, lH, H-1 GlcNAc), 4.32 (d, J = 7.7 Hz, lH, H-1 Gal), 4.06 (d, J = 3.3 Hz, lH, H-4 Gal), H-1 Gal), 4.38-4.32 (m, 2H), 4.29-4.14 (m, 2H), 4.01-3.75 (multiple 3.99 (d, J = 8.2 Hz, lH), 3.91-3.83 (m, 3H), 3.74-3.61 (m, 8H). peaks, 5H), 3.61 (bt, J = 5.6 Hz, lH, H-5 GlcN), 3.45 (m, lH, OCHz3.59-3.38 (m, 7H), 1.99 (s, 3H, NHAc), 1.98 (s, 3H, NHAc), 1.19 (t, CH3), 2.12 (s, 3H, OAc), 2.01 (s, 3H, OAc), 1.76 (s, 3H, OAc), 0.96 J = 7.7 Hz,3H, CHzCH3); 13CN M R (DzO) 6 175.0 (C-0), 174.4 (C(t, 3H, CHzCH3). 0), 102.7 (C-l), 102.4 (C-1), 101.4 (C-1), 82.1, 75.8.75.7, 73.8,73.6, Ethyl 3,6-Bis-O-(3,4,6-tri-O-acetyl-2-deoxy-2-phtha~do-~-~73.4, 69.9, 69.7, 69.6, 69.4, 68.6, 66.1, 60.7, 60.5, 55.5, 55.5, 22.2 glucopyranosyl)-2-O-benzoyl-~-~-galactopyranoside (11) and Ethyl (2C, CH&=O), 14.3 (CHIC&); MS (LSI MS+) calcd for CfiIN20163,6-Bis-O-(3,4,6-tri-O-acetyl-2-deoxy-2-ph~~do-~-~-glucopyraCs: 747.4840, found 747.4845. nosyl)-4-O-((trichloroacetyl)-N-carbamoyl)-2-ObenzoyI-&D-galacEthyl 3,6-B~s-O-[B-~-galactopyranosyl-(l-4)-2-aceta~do-2topyranoside (lla). A suspension of molecular sieves (4 A, 1 g), deoxy-~-D-glucopyranosyl]-~-~-galactopyranos~de (14). Galactocollidine (0.167 mL, 1.26 mmol), silver triflate (0.3 g, 1.16 mmol), syltransferase (Ec 2.4.1.22,6 U) and uridine 5'-diphosphogalactose 4'and diol 10 (0.71 g, 0.974 mmol) in CHIC12 (10 mL) was stirred under epimerase (EC 5.1.3.2, 8 U) were added to a solution containing sodium an argon atmosphere for 1 h. The reaction mixture was then cooled to -20 "C, and a solution of 3,4,6-tri-O-acety1-2-deoxy-2-phthalimido- cacodylate (pH 7.5, 1 M, 0.4 mL), water (3.8 mL), BSA (5% solution, 88 pL), MnClz (lM, 40pL), alkaline phosphatase (EC 3.1.3.1, lU/pL, B-D-glUCOpyranOSyl bromide (0.53 g, 1.07 m o l e ) in CHzClz (1 mL) 44pL), uridine diphosphoglucosedisodium salt (161 mg, 0.285 mmol), was added. The reaction mixture was stirred at -20 "C for 30 min and the trisaccharide 13 (70 mg, 0.1 14 "01). The reaction mixture and then allowed to warm to room temperature and filtered through a was inverted several times and then allowed to sit at room temperature Celite pad. The filtrate was diluted with ethyl acetate (100 mL), washed for 72 h. The mixture was filtered and purified by chromatography with water (50 mL), 1 M citric acid (100 mL), water (50 mL), saturated (BioGel P-2, 0.1 M N W C 0 3 ) to afford 100 mg (93%) of 14 as a NaCO3, (100 mL), water (50 mL), and saturated NaCl(50 mL), dried white solid after lyophilization: R, = 0.25 (silica, 30% 1M N&OAc/ ( N ~ I S O ~and ) , concentrated. The residue was purified by chromatog2-propanol); 'H NMR (DzO) 6 4.65 (d, J = 7.8 Hz, lH, H-1 GlcNAc), raphy (silica, 40% ethyl acetateltoluene) to afford 0.967 g (86%) of 11 4.49 (d, J = 7.6 Hz, lH, H-1 GlcNAc), 4.42 (d, J = 7.6 Hz, lH, H-1 as a white solid: Rf = 0.22 (silica, 40% ethyl acetatebenzene); 'H Gal), 4.41 (d, J = 7.8 Hz, lH, H-1 Gal), 4.30 (d, J = 7.8 Hz, lH, H-1 NMR (CDC13) 6 7.95-7.76 (bd, 3H), 7.53 (dd, J = 7.0, 1.1 Hz, 2H, bridging Gal), 4.05 (d, J = 3.2 Hz, lH, H-4 Gal), 3.99-3.43 (m, 31H), phthalimido), 7.41 (m, 2H, benzoyl), 7.35 (m, 3H, benzoyl), 7.15 (dd, 1.97 (s, 6H, OAC), 1.18 (t, 3H, CHzCH3); 13C NMR (DzO) 6 174.9 J = 7.9, 7.9 Hz, IH, phthalimido), 7.12 (dd, J = 7.9, 7.9 Hz, 2H, (C=O), 174.4 (C=O), 102.9 (C-1), 102.8 (C-1), 102.6 (C-1), 102.4 @phthalimido), 5.76 (dd, J = 9.2, 10.5 Hz, lH, H- 3 GlcN), 5.55 (dd, J anomer), 101.3 @-anomer), 82.2, 78.4, 78.1, 75.4, (2 C), 74.7, 74.6, = 9.2, 10.5 Hz, lH, H-3 GlcN), 5.47 (d, J = 8.5 Hz, lH, H-1 GlcN), 73.4, 72.5, (2 C), 72.4, 72.2, 70.1 (2 C), 69.71, 69.67, 69.4, 68.4, 68.5 5.34 (d, J = 8.5 Hz, lH, H-2 Gal), 4.38-4.06 (m, 8H), 3.94 (d, J = (2 C), 66.0, 61.0, (2 C), 60.0, 59.8, 55.3, 55.0, 22.2 (CH3C=O), 17.0 2.8 Hz, lH, H-4 Gal), 3.87 (m, 2H, H-6 Gal), 3.67 (dd, J = 3.1, 9.7 (CH3C=O), 14.3 (CH3); MS (ion spray) calcd for c3&2NZo6 938, Hz, lH, H-3 Gal), 3.54 (m, lH, H-5 GlcN), 3.49-3.42 (m, lH, H-5 found 961 (M Na+), 977 (M K+). Gal), 3.37 (m, lH, OCHICH~),3.14 (m, lH, OCHZCH~), 2.13 (s, 3H, Ethyl 3,6-Bis-O-[(ammonium5-acetamido-35-dideoxy-a-~-glycOAc), 2.09 (s, 3H, OAc), 2.03 (s, 3H, OAc), 2.01 (s, 3H, OAc), 1.85 ero-~-galacto-2-nonulopyranuronate)-(2-3)~-~-galactopyranosyl(s, 3H, OAC), 1.75 (s, 3H, OAC), 0.75 (t, J = 7.1 Hz, 3H, CHzCH3). (~-~)-~-acetamido-~-deoxy-~-~-g~ucopyranosy~]-~-~-galactopyAddition of trichloroacetyl isocyanate to the NMR sample formed ranoside (15). The N-type a(2-3)-sialyl transferase (EC 2.4.99.6, 1 compound lla and caused a shift of the 4-position hydrogen of U) was added to a solution of cytidine-5'-monophosphosialic acid (0.116 galactose to 5.39 (d, J = 2.1 Hz, 1H) ppm. g, 0.166 mmol), BSA (5% solution, 0.12 mL), sodium cacodylate (pH Ethyl 3,6-Bis-O-(3,4,6-tri-O-acetyl-2-acetamido-2-d~xy-B-~-glu6.5, lM, 1.8 mL), water (5.1 mL), MnC12 (lM, 0.6 mL), alkaline copyranosyl)-2,4-di-O-acetyl36-D-galactopyran~ide (12). A solution phosphatase (EC 3.1.3.1, lU/pL), and the pentasaccharide 14 (52 mg, of trisaccharide 11 (0.9 g, 0.785 "01) and hydrazine monohydrate 55 pmol). The reaction mixture was tipped for 4 days. Since the (1.52 mL, 31.4 m o l ) in ethanol (30 mL) was refluxed for 8 h. The reaction was not complete by TLC, addition cytidine-5'-monophosreaction mixture was concentrated and the residue was dissolved in phosialic acid (0.116 g, 0.166 mmol), alkaline phosphatase (1 U/pL, pyridine (40 mL). Acetic anhydride (20 mL) was then added and the 30 pL), MnClz (lM, 0.2 mL), and N-type a(2-3)-sialyl transferase (1 reaction mixture stirred for 24 h. The reaction mixture was concentrated U) were added and the reaction mixture tipped for another 5 days. The and the residue was dissolved in CHIC12 (100 mL). The solution was mixture was filtered and chromatographed (GioGel P-2, 0.1 M N&washed with saturated NaHC03 (100 mL). The aqueous layer was HC03) to afford 75 mg (86%) of 15 as a white solid after lyophilizaextracted again with CHzClz (50 mL), and the combined organic layers tion: Rf = 0.15 (silica, 30% 1 M NH40Acl2-propanol); 'H N M R (D20) were dried (Na~S04)and concentrated. The residue was chromato6 4.66 (d, J = 7.8 Hz, lH, H-1 GlcNAc), 4.51 (bd, 3H, H-lapos of graphed (silica, 3% MeOWethyl acetate) to afford 0.57 g (76%) of 12 GlcNAc, Gal, Gal), 4.32 (d, J = 7.8 Hz,lH, H-1 GlcNAc), 4.1-3.5 as white solid: Rf = 0.33 (silica, 5% MeOWethyl acetate); 'H NMR (m, 45H), 2.71 (dd, J = 12.3,4.3 Hz, 2H, H-3(eq) sialic acid), 1.99 (s, (CDC13) 6 5.43-5.31 (m, 3H), 5.08-4.99 (m, 3H), 4.95 (d, J = 8.1 12H, NHAc), 1.76 (dd, J = 12.5 Hz, 2H, H-3(ax) sialic acid), 1.19 (t, Hz, lH, H-1 GlcNAc), 4.77 (d, J = 8.3 Hz, lH, H-1 GlcNAc), 4.37 3H, CH2CH3); MS (ion spray) calcd for Cs8H93N4042 1520, found 1519 (d, J = 8.0 Hz, lH, H-1 Gal), 4.31 (dd, J = 1.2, 12.7 Hz, lH, CHI([M - H'I-), 759 ([M - 2H+I2-). OAc), 4.24 (dd, J = 4.2, 12.4 Hz, lH, CHzOAc), 4.13-4.05 (m, 2H), Ethyl 3,6-Bis-O-[(ammonium5-acetamido-35-dideoxy-a-~-gZyc3.89-3.78 (m, 4H), 3.73-3.64 (m, 3H), 3.59-3.43 (m, 2H), 3.41 (m, ero-~-galacto-2-non~opyranuronate)-(2-3)-~-~-galactop~anosyl1H, OC&CH3), 2.10 (s, 3H, OAc), 2.09 (s, 3H, OAc), 2.08 (s, 3H, (144)-(a-~-fucopyranosyl-( 1-3))-2-acetamid0-2-deoxy-~-~-gluOAc), 2.07 6,3H, OAc), 2.01 (s, 9H, OAc), 2.00 (s, 3H, OAc), 2.95 copyranosyl]-/?-~-galactopyranoside (6). The flucosyltransferase V (s, 3H, NHAc), 1.90 (s, 3H, NHAc), 1.17 (t, J = 7.0 Hz, 3H, CHz(100 mU attached to beads) was added to a solution containing sodium CH3). cacodylate (pH 6.5, 0.5 mL), water (4.5 mL), MnClz (lM, 0.2 mL),
+
+
Ligand Recognition by E-Selectin
J. Am. Chem. SOC.,Vol. 117, No. I , 1995 15
alkaline phosphatase (32 U), GDP-fucose disodium salt (0.11 g, 0.18 then fiitered and the filtrate concentrated. The residue was dissolved mmol), and the heptasaccharide 15 (56 mg, 36 pmol). The reaction in methanol (25 mL) and 21b crystallized by the addition of ether to mixture was tipped for 48 h. Additional transferase (100 mu) and yield 5.9 g (92%) as a white solid: Rj = 0.53 (silica, CHClfleOW MnClz (lM, 0.2 mL) were added, and the reaction mixture was tipped HzO 60/50/15); 'H N M R (DzO) 6 4.33 (d, J = 7.9 Hz, 2H, H-l), 3.92for another 4 days. The mixture was filtered and chromatographed 3.85 (m, 2H), 3.77-3.55 (m, lOH), 3.43 (m, 2H, OCWs), 1.60 (m, (BioGel P-2,O. 1 M W H C O 3 ) to afford 40 mg (60%) of 6 as a white 4H, CHz's), 1.40 (m, 2H, CH2's); MS (FAB) calcd for C16H30012 414, solid after lyophilization: Rj = 0.38 (silica, 40% 1 M NH40Ac/2found 451 (M K+)I-. propanol); 'H NMR (D20, 500 MHz) 6 5.13 (d, J = 3.5 Hz, lH, H-1 1,4-Bis((2,6-di-0-acetyl-~-~-galactopyranosyl)oxy)bu~e (22a). Fuc), 5.12 (d, J = 3.5 Hz, lH, H-1 Fuc), 4.72 (d, J = 8.0 Hz, lH, H-1 Compound 21a (6 g, 14.5 "01) was suspended in 300 mL of dry GlcNAc), 4.59-4.51 (multiple peaks, 3H, H-1's of GlcNAc, Gal and acetone, 10 mL of 2,2-dimethoxypropane, and 10 mL of dry DMF. Gal), 4.37 (d, J = 8.0 Hz, 1H H-1 bridging Gal ), 4.10 (d, J = 2.9 Hz, Five drops of concd sulfuric acid were added, and the mixture was lH, H-3 Gal), 4.10 (d, J = 2.9 Hz, lH, H-3 Gal), 4.03-3.47 (m, 54 refluxed for 3 h. Solid NaHC03 was added until neutral and the mixture H), 2.77 (dd, J = 12.4, 4.4 Hz, 2H, H-3(eq) sialic acids) 2.03 (s, 6H, filtered and concentrated. The syrupy residue was extracted with NHAc), 2.01 (s, 6H, NHAc), 1.81 (dd, J = 12.4, 12.4 Hz, 2H, H-3(m) hexane and then dissolved in 50 mL of pyridine. Acetic anhydride sialic acids), 1.24 (t, 3H, CHZCH~), 1.18 (d, J = 6.5 Hz, 6H, CH3(25 mL) was then added and the mixture stirred for 8 h. After fucose); H R M S (LSlMS-) calcd for c7&1705& (M Ht) 1811.6579, concentration, the residue was dissolved in 75 mL of CHzCl2, washed found 1811.6693. with water (50 mL), 0.1 M HCl(3 x 50 mL), water (50 mL), saturated 1,4-Bis((2,3,4,6-tetra-0-acetyl-~-~-galactopyranosyl)oxy)butae NaHCO3 (3 x 50 mL), water (50 mL), and brine (50 mL), and dried (20a). A suspension of dry molecular sieves (4 A, 5 g), CHIC12 (75 (Na2S04). Concentration afforded a viscous oil which was refluxed mL), silver carbonate (14 g, 51 mmol), and 1,4-butanediol(2.25 g, 25 for 3 h in 50 mL of acetic acid (70%). After concentration, the residue "01) was stirred under argon for 20 min. The reaction mixture was was crystallized from methanovether. Chromatography (silica, chlothen cooled to -20 "C, and a solution of 2,3,4,6-tetra-O-acetyl-u-~- rofodmethanol5:l) and recrystallization in methanoyether afforded galactopyranosyl bromide (21 g, 51 "01) in CHzClz (50 mL) was 2.3 g (28%) of 22a as white crystals: R,f = 0.38 (silica, chloroform/ added. The reaction mixture was then stirred at -20 "C overnight, methanol 4:l); 'H NMR (CDC13) 6 4.95 (t, J = 7.8, 8.1 Hz, 2H, H-2), allowed to warm to room temperature, and filtered through Celite. The 4.33 (m, 6H, H-1, H-6), 3.88 (m, 4H, H-4, OCH), 3.65 (m, 4H, H-3, filtrate was washed with water (50 mL), 0.1 M HC1 (50 mL), water H-5), 3.51 (bm, 2H, OCH), 2.85 (bs, 4H, OH), 2.12 (s, 6H, OAc), (50 mL), saturated NaHCO3 (50 mL), water (50 mL), and brine (50 2.10 (s, 6H, OAc), 1.63 (bm, CHz's). mL) and dried (Na~S04).Concentration and chromatography (silica, 1 , 5 - B i s ( ( 2 , 6 - d i - O - a c e t y l - ~ - ~ - g a l a c t o p y(22b). re ethyl acetatelhexane 1:l) 13.8 g (73%) of 20a as an amorphous foam: Compound 21b (5 g, 11.7 "01) was suspended in 300 mL of dry Rj = 0.52 (silica, ethyl acetatelhexane 2:l); 'H N M R (CDCl3) 6 5.37 acetone, 10 mL of 2,2-dimethoxypropane, and 10 mL of dry DMF. (d, J = 3.1 Hz, 2H, H-4apos). 5.17 (t, J = 7.9, 8.0 Hz, 2H, Hz's), 5.00 Five drops of concd sulfuric acid were added, and the mixture was (t, J = 3.1, 7.9 Hz, 2H, H-~'s),4.44 (d, J = 7.9 Hz, 2H, H-l), 4.10 refluxed for 3 h. Solid NaHCO3 was added until neutral, the mixture (m, 4H, H6 and H-5), 3.88 (m, 4H, H-6 and OCH), 3.45 (bm, 2H, filtered, and the filtrate concentrated. The syrupy residue was extracted 0 0 , 2.12 (s, 3H, OAc), 2.03 (s, 3H, OAc), 2.02 (s, 3H, OAc), 1.60 with hexane and then dissolved in 50 mL of pyridine. Acetic anhydride (m 4H, CH-2's); "C NMR (CDC13) 6 170.3, 170.1, 170.0, 169.3, 101.1, (25 mL) was then added and the mixture stirred for 8 h. After (C-1), 70.8, 70.5, 69.5, 68.8, 66.9, 61.1, 25.7, 20.62, 20.55, 20.47. 1,5-B~s((2,3,4,6-tetra-O-acetyl-~-~-galactopyranosy~)oxy)pen- concentration, the residue was diluted to 75 mL of CHzC12, washed with water (50 mL), 0.1 M HCl(3 x 50 mL), water (50 mL), saturated tane (20b). A suspension of dry molecular sieves (4 A, 5 g), CHzClz NaHC03 (3 x 50 mL), water (50 mL), and brine (50 mL), and dried (75 mL), silver carbdnate (14 g, 51 mmol), and 1,5-pentanediol(2.6 g, (NazS04). Concentration afforded an oil which was refluxed for 3 h 25 m o l ) was stirred under argon for 20 min. The reaction mixture in was cooled to -20 "C and a solution of 2,3,4,6-tetra-O-acetyl-a-~- 50 mL of acetic acid (70%). Concentration and crystallization from methanovether after a solid which was chromatographed (silica, galactopyranosyl bromide (21 g, 51 "01) in CHIC12 (50 mL) added. chlorofodmethanol7: 1-5: 1) and then recrystallization in methanol/ This mixture was stirred at -20 "C overnight and then allowed to warm ether afforded 2.5 g (38%) of 22b as white crystals: Rj= 0.49 (silica, to room temperature before filtering through Celite. The filtrate was chlorofodmethanol 4:l); 'H NMR (CDC13) 6 4.92 (d, J = 8.0, 8.2 washed with water (50 mL), 0.1 M HC1 (50 mL), water (50 mL), Hz, 2H, H-2), 4.30 (m, 6H, H-1, H-6), 3.89 (d, J = 3.1 Hz, 2H, H-4), saturated NaHC03 (50 mL), water (50 mL), and brine (50 mL) and 3.82 (m, 2H, OCH), 3.65 (m, 4H, H-3, H-5), 3.46 (m, 2H, OCH), 3.32 dried (NazS04). Concentration and chromatography (silica, ethyl (bs, 4H, OH), 2.10 (s, 3H, OAc), 2.07 (s, 3H, OAc), 1.55 (m, 4H, acetatelhexane 1:l) afforded 13 g (68%) of 20b as an amorphous CHZ),1.36 (m, 2H, CHI). I3C NMR (CDC13) 6 171.4, 171.1 (C-0), foam: Rj = 0.5 (silica, ethyl acetatelhexane 2: 1); 'H NMR (CDC13) 6 100.8 (C-l), 72.9, 72.3, 72.0, 69.4, 68.8, 62.8, 28.9, 22.2, 21.0, 20.8. 5.37 (d, J = 3.0 Hz, 2H, H-4), 5.17 (t, J = 7.9, 8.1 Hz, 2H, H-~'s),
+
+
5.00(t,J=3.0,7.9H~,2H,H-3),4.44(d,J=8.0H~,2H,H-l),4.12 1,4-Bis((3,4,6-tri-0-acetyl-2-deoxy-2-ph~~do-~-~-~ucopyra(m, 4H, H-6 and H-5), 3.85 (m, 4H, H-6 and OCH), 3.49 (bm, 2H, nosyl-(1-3)-2,6-di-0-acetyl-~-~-galactopyranosyl)oxy)butane (23). OCH), 2.14 (s, 3H, OAc), 2.04 (s, 3H, OAc), 2.03 (s, 3H, OAc), 1.61 A suspension of molecular sieves (4 A, 1 g), CHIC12 (30 mL), collidine (m, 4H, CHI), 1.30 (m, 2H, CHZ);I3C NMR (CDC13) 6 170.3, 170.2, (0.3 mL, 2.3 mmol), silver triflate (0.57 g, 2.2 mol), and compound 66.9, 61.1,29.0,22.1, 170.1, 169.3, 101.2 (C-1),70.8,70.5,69.9,68.8, 22a (0.5 g, 0.876 "01) was stirred under argon for 1 h. The reaction 20. mixture was then cooled to -20 "C, and a solution of 2-deoxy-2phthalimido-3,4,6-triri-O-acetyl-~-~-glucopyranosyl bromide ( 1.06 g, 1,4-Bis-(~-~-galactopyranosyl)oxy)butane (21a). Sodium meth2.14 "01) CHzClz (20 mL) was added. The reaction mixture was oxide (100 mg) was added to a solution of compound 20a (13 g, 17.3 ~
"01) in MeOH (80 mL), and the reaction mixture was stirred for 2 h. The prewashed Biogel AG 50W-X8 (hydrogen form) was added to the mixture until neutral. The reaction mixture was filtered and concentrated. The residue was dissolved in methanol (25 mL) and 21a crystallized by the addition of ether to yield 6.6 g (92%) as a white solid: Rj = 0.52 (silica, CHC13/MeOH/H2060/50/15); 'H N M R (CD3OD) 6 4.19 (d, J = 7.0 Hz, 2H, H-l), 3.92-3.29 (m, 24H). 1.70 (m, 4H, CHz's); MS (FAB) calcd for c1&& 414, found 437 (M Na+), 453 (M K+). l~-(Bis-((B-D-galactopyranosyl)oxy)pentae (21b). Sodium methoxide (100 mg) was added to a solution of compound 20b (11.5 g, 15 "01) in MeOH (80 mL), and the reaction mixture was stirred for 2 h. Then prewashed Biogel AG 50W-X8 (hydrogen form) was added to the mixture until the pH was neutral. The reaction mixture was
+
+
stirred at -20 "C for 30 min, warmed to room temperature overnight, and filtered through Celite. The filtrate was diluted with dichloromethane (100 mL), washed with water (50 mL), 0.1 M HCl (100 mL), water (50 mL), saturated NaHC03 (100 mL), water (50 mL), and brine (50 mL), and dried (Na~S04).Concentration and chromatography (silica, acetone/toluene 1:2) afforded 0.650 (52%) of 23 as a white solid Rj= 0.25 (silica, toluene/acetone 2:l); 'H NMR (CDC13) 6 7.84 (m, 4H, Phth), 7.76 (m, 4H, Phth), 5.74 (dd, J = 10.6, 10.4 Hz, 2H, H-3 Glc), 5.50 (d, J = 8.3 Hz, 2H, H-1), 5.16 (dd, J = 8.3, 8.5 Hz, 4H, H-4 Glc, H-3 Glc), 4.92 (dd, J = 8.2, 8.2 Hz, 2H, H-2 Gal), 4.384.22(m, lOH),4.15(d,J=8.lHz,2H,H-lGal),3.99-3.84(m,4H), 3.71-3.58 (m, 4H), 3.28 (m, 2H, OCH), 2.11 (s, 12H, OAc), 2.07 (s, 6H, OAc), 2.04 (12H, OAc), 1.44 (m, 4H, CHZ);MS (ion spray) calcd for C&72N2034 417, found 1439 (M Na+)-, 1455 (M K+)I-.
+
+
76 J. Am. Chem. Soc., Vol. 117, No. I, 1995
DeFrees et al.
Acetylation of compound 23 with acetic anhydride in pyridineL5 12H, OAc), 2.06 (s, 12H, OAc), 2.04 (s, 6H, OAc), 1.96 (s, 6H, OAc), caused a shift of the 4-H of the galactoses to 5.35 (d, J = 3.5 Hz, 2H) 1.58-1.46 (m, 4H, CH2), 1.33-1.25 (m, 2H, CHz). 1,4-Bis((2-acetamido-2-deoxy-~-~-glucopyranosyl-( 1-3)-B-~-gaPPm. 1actopyranosyl)oxy)butane (26). Sodium methoxide (75 mg) was 1,4-Bis((2,3,4,6-tetra-O-acetyl-B-~-galac~pyranosyl-( 1-4)-3,4,6in MeOH added to a solution of compound 25 (40 mg, 0.03 "01) tri-O-acetyl-2-deoxy-2-phthalimido-~-~-glucopyranosyl-( 1-3)-2,6(20 mL). The reaction was stirred for 20 h during which time a di-O-aCetyl-B-D-galaC~pyranOSyl)OXy)pentane(24). A suspension precipitate formed. Water (-5 mL) was added until all of the solid of molecular sieves (4 A, 1 g), CHzClz (30 mL), collidine (0.27 mL, 2 had dissolved, and then prewashed Biogel AG 50W-X8 (hydrogen form) mmol), silver triflate (0.53 g, 2 mmol), and 22b (0.4 g, 0.68 "01) was added until neutral. The reaction mixture was filtered and was stirred under argon for 1 h. The reaction mixture was then cooled concentrated. The residue was dissolved in water (10 mL) and filtered to -20 OC and a solution of 2,3,4,6-tetra-~-acety~-~-~-galactopyranosy~through a C-18 sep peak (Whatman) and the fiitrate concentrated to (l-4)-2-deoxy-2-phth~do-3,6-di-O-acetyl-~-~-glucopyranosylchloyield 24 mg (96%) of 26 as a white solid: Rf = 0.15 (silica, CHC13/ ride (1.3 g, 1.7 "01) in CHzClz (20 mL) was added. The reaction MeOWHZO 60/50/15); IH N M R (DzO) 6 4.69 (d, J = 7.4 Hz, 2H, mixture was stirred at -20 "C for 30 min, warmed to room temperature H-1 Glc),4.37(d,J=7.9Hz,2H,H-l Gal),4.12(d,J=3.0Hz,2H, overnight and filtered through Celite. The filtrate was diluted with 4H), 3.80-3.44 (m, 20H), 2.02 (s, 6H, H-4 Gal), 3.95-3.87 (h, dichloromethane (100 mL), washed with water (50 mL), 0.1 M HC1 NHAc), 1.69 (bm, 4H, CHz). (100 mL), water (50 mL), saturated NaHC03 (100 mL), water (50 mL), 1,5-Bis((/?-~-galactopyranosyl-( 1-4)-2-acetamid0-2-deoxy-B-~and brine (50 mL), and dried (Na2SO4). Concentration and chromaglucopyranosyl-(l-3)-~-~-galactopyranosyl)oxy)pentane(31). Sotography (silica, acetone/toluene 1:1,5) afforded 0.550 g (40%) of 24 dium methoxide (0.1 g) was added to a solution of compound 30 (0.1 as a white solid: Rf = 0.38 (silica, toluene/acetone 1:l); 'H N M R in MeOH (20 mL). The reaction mixture was stirred g, 0.05 "01) (CDC13) 6 7.84 (m, 4H, Phth), 7.76 (m, 4H, Phth), 5.69 (dd, J = 10.2, for 20 h during which time a precipitate formed. Water (-5 mL) was 8.5 Hz, 2H, H-3 Glc), 5.49 (d, J = 8.4 Hz, 2H, H-1 Glc), 5.31 (3, J added until the solid had dissolved, and then prewashed Biogel AG 3.2 Hz, 2H, H-4 Gal), 5.12 (dd, J = 8.0, 7.8 Hz, 2H, H-2 Gal), 4.9950W-X8 (hydrogen form) was added to the reaction mixture until 4.87 (m, 4H, H-2 Gal, H-3 Gal'), 4.62-4.55 (m, 4H, H-6 Gal',H-5 neutral. The reaction mixture was filtered, the filtrate concentrated, Gal'), 4.32-4.30 (d, J = 8.2 Hz, 2H, H-1 Gal'), 4.28-4.24 (m, 4H, and the residue dissolved in water (10 mL). Filtration through a C-18 H-2 Glc), 4.21 (d, J = 2.9 Hz, 2H, H-4 Gal), 4.18-3.94 (m, lOH), sep pack (Whatman) and concentration afforded 56 mg (96%) of 31 as 3.92-3.76 (m, 6H), 3.70-3.63 (m, 2H, OCH), 2.14(s, 6H, OAc), 2.13 a white solid Rf= 0.23 (silica, CHClmeOWHZO 60/50.15); 'H Nh4R (s, 6H, OAc), 2.07 (s, 6H, OAc), 2.06 (s, 6H, OAc), 2.03 (s, 12H, (D20)6 4.65 (d, J = 7.8 Hz, 2H, H-1 Glc), 4.42 (d, J = 7.7 Hz, 2H, OAc), 1.96 (s, 6H, OAc), 1.90 (s, 6H, OAc), 1.44-1.30 (m, 4H, CHZ), H-1 Gal'), 4.32 (d, J = 7.9 Hz, 2H, H-1 Gal), 4.08 (d, J = 3.0 Hz, 2H, 1.25-1.12 (m, 2H, CHz); MS (ion spray) calcd for C S ~ H I I ~ O & 2008, H-4 Gal), 3.95-3.45 (m, 38H), 1.97 (s, 6H, NHAc), 1.64-1.55 (m, found 2141 (M Na+), 2139 (M Na+)2-. 4H, CHZ). 1.39-1.37 (m, 2H, CHz). AcetylationL5of compound 24 with acetic anhydride in pyridine 1,4-Bis((B-~-galactopyranosyl-( 1-4)-2-acetamid0-2-deoxy-/%~caused a shift of the galactose H-4 to 5.33 (d, J = 2.0 Hz, 2H) ppm. g~ucopyranosy~-(l-~)-~-~-galactopyranosyl)oxy)butane (27). Ga1,4-Bis((2-acetamido-3,4,6-tri-~-acetyl-2-deoxy-~-~-glucopyranolactosyltransferase (1 U)and uridine 5'diphosphogalactose 4'-epimerase syl-(~-~)-~,~,6-t~-~-acetyl-~-~-galactopyranosyl)oxy)bu~e (25). (3 U) were added to a solution containing solution cacodylate (pH 7.5, A solution of compound 23 (0.1 g, 0.067 mmol), hydrazine monohy0.2 M, 0.4 mL), water (1 mL), BSA (5% solution, 20 pL), MnClz (lM, drate (1 mL, 20.6 mmol), and ethanol (20 mL) was refluxed for 8 h. lOpL), alkaline phosphatase (1 U/pL, 10pL), uridine diphosphoglucose The reaction mixture was concentrated and the residue dissolved in disodium salt (28.3 mg, 0.05 "01, 2.5 eq), and compound 26 (17 pyridine (15 mL). Acetic anhydride (7 mL) was added, and the mixture mg, 0.02 "01). The reaction mixture was inverted several times and was stirred for 12 h and then concentrated. The residue was dissolved allowed to sit at room temperature for 72 h. Filtration and chromain CHzClz (100 mL) and washed with saturated NaHC03 (100 mL). tography (Biogel P-4, 0.1 M W H C 0 3 ) afforded 21 mg (91%) of 27 The aqueous layer was extracted with CHzClz (50 mL), and the as a white solid after lyophilization: Rf = 0.08 (silica, CHClmeOW combined organic layers were dried (NazS04). Concentration and HzO 60/50/15); 'H NmR (DzO) 6 4.70 (d, J = 7.8 Hz, 2H, H-1 Glc), chromatography (silica, toluene/acetone 1:l) afforded 50 mg (57%) of 4.47(d,J=7.6Hz,2H,H-lGal'),4.37(d,J=8.0H~,2H,H-lGal), 25 as white solid contaminated with phthalimido hydrazide (-10% by 4.13 (d, J = 3.0 Hz, 2H, H-4 Gal), 3.92-3.53 (m, 38H), 2.02 (s, 6H, 'H NMR), Rf = 0.2 (silica, toluenelacetone 1:l). This product was NHAc), 1.69 (bm, 4H, CHz); MS (ion spray) calcd for C& . +&&NZ used directly for the next step: 'H NMR (CDC13) 6 5.54 (dd, J = 7.5, 1145, found 1167 (M Na+)+. 8.2 Hz, 2H), 5.36 (d, J = 3.2 Hz, 2H, H-4 Gal), 5.13-4.99 (m, 6H), 1,4-Bis((ammonium 5-acetamido-3,5-dideoxy-a-~-gZycero-~-ga4.36-4.03 (m, 5H), 3.89-3.76 (m, 6H), 3.71-3.63 (m, 4H), 3.50lacto--nonulopyranosuronate)-(2-3)-~-~-galactopyranosyl-( 1-4)3.44 (m, 2H, OCH), 2.12 (s, 3H, OAc), 2.11 (s, 3H, OAc), 2.09 (s, 2-acetamido-2-deoxy-~-~-glucopyranosyl-( 1-3)-B-~-gaIactopyra3H, OAc), 2.01 (s, 3H, OAc), 1.91 (s, 6H, OAc), 1.71-1.62 (m, 4H, nosy1)oxy)butane (28). The a(2-3)-sialyl transferase (0.1 U) was CH3. added to a solution of cytidine 5'-monophosphate-~ialic acid (27.5 1~-Bis((2,3,4,6-tetra-O-acetyl-~-~-galactopyranosyl-( 1-4)-2-acmg, 0.04 mmol), BSA (5% solution, 10 pL), sodium cacodylate (pH etamido-3,4,6-t~-O-acetyl-2-deoxy?g-~-glucop~anosyl-( 1-3)-2,3,66.5, 0.2 M, 1.25 mL), water (1 mL), Triton (0.2 M, 25 pL), alkaline tri-O-aCetyl-B-D-galaCtOpyranOSyl)OXy)pentane(30). A solution of phosphatase (1 U/pL, 24 pL), and compound 27 (15 mg, 13 pmol). compound 24 (0.275 g, 0.137 mmol), hydrazine monohydrate (1 mL, The reaction mixture was incubated at 37 "C for 2 days. Filtration 20.6 mmol), and ethanol (20 mL) was refluxed for 8 h. The reaction and chromatography (Biogel P-4, 0.1 M m C 0 3 ) afforded 21 mg mixture was concentrated and the residue dissolved in pyridine (15 (93%) of 28 as a white solid after lyophilization: Rf = 0.53 (silica, mL). Acetic anhydride (7 mL) was then added and the reaction mixture CHCl3lMeOWl M W O A c 1/4/1); IH N M R (DzO) 6 4.65 (d, J = stirred for 12 h. After concentration the residue was dissolved in CH27.8 Hz, 2H, H-1 Glc), 4.50 (d, J = 7.8 Hz, 2H, H-1 Gal'), 4.33 (d, J Clz (100 mL) and washed with saturated NaHC03 (100 mL). The = 7.9 Hz, 2H, H-1 Gal), 4.08 (d, J = 2.8 Hz, 2H, H-4 Gal'), 4.06 (dd, aqueous layer was extracted again with CHZC12 (50 mL), and the J = 2.8, 10.1 Hz, 2H, H-3 Gal'), 3.92-3.45 (m, 50H), 2.71 (dd, J = combined organic layers were dried (NaZS04). Concentration and 12.3, 4.4 Hz, 2H, H-3 SA), 1.97 (s, 12H, NHAc), 1.75 (dd, J = 12.3 chromatography (silica, toluenelacetone 1:1) afforded 227 mg (86%) , 12.1 Hz, 2H, H-3 SA), 1.64 (bm, 4H, CHZ);MS (ion spray) calcd for of 30 as a white solid containing some phthalamido hydrazide as a Cd110048N4 1728, found 1726 (M)'-. contaminant (-15% by 'H NMR). This product was used without 1,5-Bis(((ammonium 5-acetamido-35-dideoxy-a-~-gZycero-~-gafurther purification: Rf = 0.34 (silica, toluenelacetone 1:l); lH N M R lacto-nonulopyranosuronate)-(2-3)-/3-~-galactopyranosyl-(1-4)-2(CDC13) 6 5.41 (dd, J = 8.6, 8.6 Hz, 2H, H-3 Glc), 5.35 (bs, 2H), 5.21 acetamiao-zdeoxy-~-~~ucopyranosyl-( 143)-/?-~-galactopyy1)(dd, J = 9.4, 9.4 Hz, 2H), 5.14-4.94 (m, 4H), 4.75-4.69 (m, 2H), oxy)pentane (32). The a(2-3)-sialyl transferase (0.135 U) was added 4.53(d,J=7.9H~,2H,H-lGlc),4.33(d,J=8.1H~,2H,H-lGal), to a solution of cytidine 5'-monophosphate-sialic acid (54.5 mg, 0.078 4.18-3.96 (m, lOH), 3.88-3.75 (m, 8H), 3.58-3.50 (m, 4H), 3.46mmol), BSA (5% solution, 5 pL), sodium cacodylate (pH 6.5,0.2 M, 3.41 (m, 2H, OCH), 2.15 (s, 12H, OAc), 2.11 (s, 6H, OAc), 2.07 (s, 2.56 mL), water (1.75 mL), Triton (0.2 M, 51.7 pL), alkaline
+
+
+
Ligand Recognition by E-Selectin
J. Am. Chem. SOC., Vol. 117, No. 1, 1995 I1
phosphatase (1 U/pL, 27 pL), and compound 31 (30 mg, 26 pmol). 5-(Methoxycarbonyl)pentyl2,6-Bis-O-[(a"onium 5-acetamidoThe reaction mixture was incubated at 37 "C for 2 days. Filtration 3,5-dideoxy-a-~g~cero-D-ga~~-2-non~opyranosuronate)-(2-3)and chromatography (Biogel P-4, 0.1 M m H C 0 3 ) afforded 44 mg B-~palactopyranosyl-(l-4~~a~~d~~eoxy-B-~~u~pyranosyl](93%) of 32 as a white solid after lyophilization: Rf = 0.53 (silica, B-D-galactopyranoside(35). Yield 18 mg (71%) of 35 a a white CHCl&leOH/l M m 0 A c 1:4:1); IH NMR (DzO) 6 4.70 (d, J = solid: Rf= 0.16 (silica, 30% 1 M N€LOAc/2-propanol); 'H NMR ( 4 0 ) 6.8 Hz, 2H, H-1 Glc), 4.54 (d, J = 7.8 Hz, 2H, H-1 Gal'), 4.36 (d, J 6 4.66 (bd, lH, j3-anomer Glc), 4.53 (bd, 3H, j3-anomers of Glc, Gal, = 7.9 Hz, 2H, H-1 Gal), 4.12 (d, J = 2.9 Hz, 2H, H-4 Gal'), 4.09 (dd, Gal), 4.39 (d, J = 7.4 Hz, lH, j3-anomer, bridging Gal), 4.09 (dd, J = J = 3.0, 10.3 Hz, 2H, H-3 Glc), 3.95-3.50 (m, 50H), 2.76 (dd, J = 2.4 Hz, lH, H-3 Gal), 4.06 (dd, J = 2.4 Hz, lH, H-3 Gal), 3.98-3.40 12.3, 4.4 Hz, 2H, H-3 SA), 2.02 (s, 12H, NHAc), 1.79 (dd, J = 8.1, (m, 47H), 2.71 (dd, J = 12.3,4.3 Hz, 2H, H-3(eq) sialic acid), 2.37 (t, 12.1 Hz, 2H, H-3 SA), 1.67-1.62 (m, 4H, CH2), 1.44-1.33 (m, 2H, J = 7.4 Hz, 2H, CHzCOOMe), 1.98 (s, 9H, NHAc), 1.97 (s, 3H, CHz): MS (ion spray) calcd for C67H112048N41742, found 870 (M)z-. NHAc), 1.76 (dd, J = 12.5 Hz, 2H, H-3(ax) sialic acid), 1.64-1.54 1,4-Bis(ammonium 5-acetamido-3,5-dideoxy-a-~-glycero-~-ga-(m, 4H, CHz alkyl), 1.37-1.32 (m, 2H, CH2 alkyl); MS (ion spray, lacto-nonulopyranosurodcacid)-(2-3)-/?-~galactopyranosyl-(l-4)positive ion) calcd for C~~HIOZN~OM 1619, found 1619 (M+), 1641 ([M+ (6-deoxy-a-~-ga~actopyranosyl-(l~3))-2-acetamido-2-deoxy-~-~Na+])I-. g~ucopyranosyl-(~-~)-~-~-galactopyranosyl)oxy)butane (29). The 5-(Methoxycarbonyl)pentyl3,4-Bis-O-[(ammonium5-acetamidofucosyltransferase V (80 mU attached to beads) was added to a solution 3,~-dideoxy-a-D-g~cero-D-ga~to-2-nonulopyranosuronate)-(2-3)containing sodium cacodylate (pH 6.5,0.2 M, 1.1 mL), water (1.1 mL), ~ - ~ ~ ~ p y r a n o s y l - ( l - 4 ) - 2 a c e ~ d ~ ~ e o ~ ~ ~ MnC12 (1 M, 0.2 mL), alkaline phosphatase (40 U), GDP-fucose B-D-gdactopyranoside(36). Yield 18 mg (71%) of 36 as a white disodium salt (15 mg, 0.025 mmol), and compound 28 (17.3 mg, 10 solid Rf= 0.21 (silica, 30% 1M WOAd2-propanol); 'H NMR (DzO) pmol). The reaction mixture was tipped for 48 h, filtered, and 6 4.58 (d, J = 8.4 Hz, lH, p-anomer Glc), 4.50 (m. 3H, p-anomers of chromatographed (Biogel P-4, 0.1 M NH4HCO3) to afford 18.8 mg Glc, Gal, Gal), 4.30 (dd, J = 2.4 Hz, lH, H-3 Gal), 4.06 (dd, J = 2.4 (93%) of 29 as a white solid after lyophilization: Rf = 0.24 (silica, Hz, lH, H-3 Gal), 3.95-3.43 (m, 45 H), 3.31 (t, J = 8.0 Hz, lH, OCH 2-propanoV1 M NH40Ac 211); IH NMR (DzO) 6 5.06 (d, J = 3.6 Hz, alkyl), 2.70 (dd, J = 12.3, 4.3 Hz, 2H, H-3(eq) sialic acid), 2.34 (t, J 2H, H-1 Fuc), 4.65 (d, J = 7.9 Hz, 2H, H-1 Glc), 4.47 (d, J = 8.2 Hz, = 7.3 Hz, 2H, CH2COOMe), 1.98 (s, 3H, NHAc), 1.97 (s, 9H, NHAc), 2H,H-lGal'),4.32(d,J=7.8Hz,2H,H-lGal),4.08(d,J=2.3Hz,1.74 (dd, J = 12.1 Hz, 2H, H-3(ax) sialic acid), 1.59-1.52 (m, 4H, 2H, H-4 Gal'), 4.03 (dd, 2H, H-3 Gal'), 3.94-3.44 (m, 56H), 2.71 (dd, CHz alkyl), 1.34-1.28 (m, 2H, CHI alkyl); MS (ion spray, positive J = 12.3, 4.4 Hz, 2H, H-3 SA), 1.97 (s, 12H, NHAc), 1.73 (dd, J = ion) calcdfor C36H102N4044 1619, found 1619 (M+), 1641 ([M+ Na+] 12.1 Hz, 2H, H-3 SA), 1.65 (bs, 4H, CHz), 1.10 (d, J = 6.3 Hz, CH3H'). Fuc); MS (ion spray) calcd for C78H130056N4 2020, found 1008 (M)l/z2-. 5-(Methoxycarbonyl)pentyl2,3-Bis-O-[(ammonium5-acetamido1,5-Bis(((ammonium 5-acetamido-3,5-dideoxy-a-~-glycero-~-ga3,5-dideoxy-a-D-glycero-D-galacto-2-non~opyranosuonate)-(2-3)lacto-nonulopyranosuodcacid)-(2-3)-B-~-g~ctopyran~yl-( 1-4)B-~-galactopyranql-( 1-4)-2ace~d~2deoxy~oxy-B-DglucopyranosylI(a-L-fucopyranosyl-(l-3)-2-acetamid~2-deoxy-~-~-~ucopyranql- /h-galactopyranoside (37). Yield 16 mg (64") of 37 as a white (1-3)-B-~-galactopyranosyl)oxy)pentane (33). The fucosyltransferase solid: Rf = 0.29 (silica, 30% 1 M NI&OAc/2-propanol); IH NMR (D20) V (100 m u attached to beads) was added to a solution containing 6 4.66-4.49 (multiple peaks, 5H, p-anomers Glc, Glc, Gal, Gal, sodium cacodylate (pH 6.5, 0.2 M, 2 mL), water (2 mL), MnClz (lM, bridging Gal), 4.09 (dd, J = 2.2 Hz, lH, H-3 Gal), 4.06 (dd, J = 2.7 0.2 mL), alkaline phosphatase (60 U), GDP-fucose disodium salt (25 Hz, lH, H-3 Gal), 3.98-3.41 (m, 46H). 2.72 (dd, J = 12.3, 4.3 Hz, mg, 0.042 mmol), and compound 31 (30 mg, 17 pmol). The reaction 2H, H-3(eq) sialic acid), 2.38 (t, J = 7.4 Hz, 2H, C&COOMe), 2.07 mixture was tipped for 48 h. Filtration and chromatography (Biogel (s, 3H, NHAc), 2.03 (s, 3H, NHAc), 1.99 (s, 6H, NHAc), 1.76 (dd, J P-4, 0.1 M N m C 0 3 ) afforded 31 mg (93%) of 33 as white solid = 12.1 Hz, 2H, H-3(ax) sialic acid), 1.64-1.55 (m, 4H, CH2 alkyl), after lyophilization: Rf = 0.24 (silica, 2-propanoV1 M N b O A c 2/1); 1.39-1.34 (m, 2H, CHz alkyl); MS (ion spray, positive ion) calcd for 'H NMR (Dz0) 6 5.11 (d, J = 3.4 Hz, 2H, H-1 Fuc), 4.70 (d, J = 8.2 C,~~HIOZN~OU 1619, found 1619 (M+), 1641 ([M+ Na+])'-. H~,2H,H-lGlc),4.51(d,J=8.2H~,2H,H-lGal'),4.32(d,J=7.8 Enzymatic Fucosylation of 34-37. 5-(Methoxycarbony1)pentyl Hz,2H,H-l Gal),4.14(d,J=2.1 H~,2H,H-4Gal'),4.07(dd,J= 4,6-Bk-O-[(ammonium 5-acetamido-3,5-dideoxy-a-~-g~ycero-~-ga3.0, 9.7 Hz, 2H, H-4 Gal'), 3.98-3.48 (m, 56H), 2.76 (dd, J = 12.3 ~to-2-non~opyranouronate)-(2-3)-b-~-galactopyran~yl-( 1-4)Hz, 4.0 Hz, 2H, H-3 SA), 2.01 (s, 12H, NHAc), 1.79 (dd, J = 12.1, (a-~-fucopyranosyl-(l-3)-2acetamido-2-d~xy-~-~-~ucop~osyl]12.1 Hz, 2H, H-3 SA), 1.64 (bm, 4H, CH2), 1.41 (bm, 2H, CH2), 1.15 B-D-galactopyranoside(16). The fucosyltransferase V (200 mU (d, J = 5.7 Hz, 6H, CH3-Fuc); MS (ion spray) calcd for C7&hO& attached to beads) was added to a solution containing sodium cacodylate 2034, found 2032 (M)2-, 1015 (M)I&. (pH 6.5, 1 M, 0.3 mL), water (1.8 mL), MnClz (1 M, 0.1 mL), alkaline phosphatase (32 U), GDP-fucose disodium salt (34 mg, 55 pmol), and Enzymatic Sialylation for the Synthesis of 34-37. 5-(Methylcarbonyl)pentyl4,6-Bk-O-[(ammonium5-acetamido-3,5-dide-a-~34 (16 mg, 9.6 pmol). The reaction mixture was tipped for 5 days. glycero-~-galacto-2-nonulopyranosuronate-(2-3)-~-~-galactopy- Filtration and chromatography (Biogel P-2, 0.1 M NH&IC03) of the r a n o s y ~ - ( ~ - ~ ) - ~ - a c e ~ d o - ~ - d e o x y - ~ - ~ - ~ u c o p y r ~ o s y l ] -filtrate ~ - ~ - gafford ~ a c - 17 mg (98%) of 16 as white solid after lyophilization: topyranoside (32). The N-type a(2-3)-sialyl transferase (EC 2.4.99.6, Rf = 0.56 (silica, 40% 1 M NH40Acl2-propanol); 'H NMR (D20) 6 5.07 (d, J = 3.5 Hz, lH, a-anomer Fuc), 5.05 (d, J = 3.5 Hz, lH, 1 U) was added to a solution of cytidine-5'-monophosphate-sialic acid (33 mg, 0.048 mmol), BSA (5% solution, 0.05 mL), sodium cacodylate a-anomer Fuc), 4.63 (d, J = 7.8 Hz, 1 H, p-anomer Glc), 4.53-4.47 (pH 6.5, 1 M, 0.9 mL), water (2.6 mL, MnC12 (1 M, 0.2 mL), alkaline (bd, 3H, j3-anomers Glc, Gal and Gal), 4.02 (d, J = 2.5 Hz, lH, H-3 phosphatase (EC 3.1.3.1, 1 U/pL, 30 pL), and 5-(methoxycarbony1)Gal), 3.99-3.44 (m, 42 H), 3.27 (t, J = 8.8 Hz, lH, OCH alkyl), 2.71 1-4)-2-acetamid0-2-deoxypentyl 4,6-bis-O- ~-D-galactopyranosyl-( (dd, J = 12.4, 4.4 Hz, 2H, H-3(eq) sialic acids), 2.36 (t, J = 7.4 Hz, ~-D-glucopyranosyl]-~-D-gala~topyranoside'~ (16 mg, 16 pmol). The 2H, CHz alkyl), 1.98 (s, 9 H, NHAc), 1.95 (s, 3H, NHAc), 1.74 (dd, J = 12.4, 12.4 Hz, 2 H, H-3(ax) sialic acids), 1.60-1.53 (m. 4H, CHz reaction mixture was allowed to stand for 4 days. Filtration and chromatography (Biogel P-2,0.1 M NKHC03) of the filtrate afforded alkyl),1.38-1.31(m,2H,CH~alkyl),1.11(d,J=6.5Hz,6H,CH~18 mg (71%) of 32 as white solid after lyophilization: R f = 0.21 (silica, fucose); MS (ion spray) calcd for C ~ ~ H ~ Z Z1912, N~O found ~ Z 1912 (M+), 30% 1 M NI&OAc/2-propanol); 'H NMR (DzO) 6 4.68 (d, J = 7.5 955 (M+ - 2NH4). Hz, lH, B-anomer Glc), 4.53 (bd, 3H, p-anomers of Glc, Gal, Gal), 5-(Methoxycarbonyl)pentyl2,6-Bis-O-[(ammonium5-acetamido4.29 (d, J = 8.0 Hz, lH, p-anomer, bridging Gal), 4.08 (dd, J = 2.8 3,5-dideoxy-a-D-g~ycero-D-galacto-2-nonulopyranurona~)-(2-3)-~Hz, lH, H-3 Gal), 4.05 (dd, J = 2.8 Hz, lH, H-3 Gal), 3.97-3.49 (m, D-galactopyranosyl-( 1-4)-(6-deoxy-a-~-galactopyranosyl-(1-3)-246H), 3.31 (dd, J = 7.8, 9.6 Hz, lH, OCH alkyl), 2.73 (dd, J = 12.3, ace~do-2-deoxy-~-~-glucopyr~osyl]-~-~-galactopyranoside (17). 4.3 Hz,2H, H-3(eq) sialic acid), 2.39 (t, J = 7.4 Hz,2H, CH2COOMe), Yield 18 mg (85%) of 17 as white solid after lyophilization: Rf = 2.02 (s, 3H, NHAc), 2.01 (s, 6H, NHAc), 1.99 (s, 3H, NHAc), 1.78 0.41 (silica, 40% 1 M NbOAc/2-propanol); 'H NMR (D20) 6 5.07 (dd, J = 12.5 Hz, 2H, H-3(ax) sialic acid), 1.64-1.58 (m, 4H, CHZ (d, J = 3.5 Hz, lH, a-anomer Fuc), 5.05 (bd, 2H, a-anomers Fuc), alkyl), 1.39 (m, 2H, CHz alkyl); MS (ion spray positive ion) calcd for 4.64 (d, J = 8.4 Hz, lH, /3-anomer Glc), 4.51-4.44 (multiple peaks, C63H102N40~1619, found 1641 [(M+ -k Na+])'-. 3H, p-anomers Glc, Gal and Gal), 4.38 (d, J = 7.4 Hz, lH, B-anomer
+
+
+
+
1 8 J. Am. Chem. Soc., Vol. 117, No.
I, 1995
DeFrees et al.
codigested with Cla I and Xho I restriction endonucleases and subcloned bridging Gal), 4.06 (d, J = 2.7 Hz, lH, H-3 Gal), 4.02 (d, J = 2.3 Hz, into the Cla I and Xho I restriction sites of the cloning vector pBluescript lH, H-3 Gal), 3.99-3.38 (m, 41 H), 2.71 (dd, J = 12.4, 4.4 Hz, 2H, 11, generating pBSII-sol-E-selectin. Soluble E-selectin is 527 amino H-3(eq) sialic acids), 3.28 (t, J = 7.4 Hz, 2H, CHI alkyl), 1.98 (s, 9H, acids in length and contains 11 potential N-glycosylation sites. A 1.67 NHAc), 1.96 (s, 3H, NHAc), 1.74 (dd, J = 12.4, 12.4 Hz, 2H, H-3(=) Kbp DNA fragment containing the soluble E-selectin cDNA was sialic acids), 1.62-1.55 (m, 4H, CH2 alkyl), 1.37-1.32 (m, 2H, CH2 isolated from pBSII-sol-E-selectin and sub-cloned into the EcoRV and alkyl), 1.11 (d, J = 6.5 Hz, 6H, CH3-fucose); MS (ion spray) calcd for Xho I sites of the expression vector pCDNAI generating pCDNAIC75H122N4052 1912, found 1912 (M+), 955. sol-E-selectin. 5-(Methoxycarbonyl)pentyl3,4-Bis-O[(ammonium 5-acetamidoGeneration of a Stable Cell Line Secretin Sol-E-selectin. pCD~,~-dideoxy-a-D-g~cero-D-gu~to-~-non~opyranosy~onat)-(~~~)B)-~-gdactopyranosyI-(l-4)-(6-deoxy-a-~-gdactopyran~yl-(l~3)- NAI-sol-E-selectin was cotransfected with pSV2-ne0, via the calcium phosphate technique?O into 293 cells. At 48 h post-transfection, the (19). 2-acetamiaO-2-deoXy-B-o-plucopyranosyl]-fl-~g~~p~~de transfected 293 cells were trypsinized and plated into DMEM, 10% The fucosyl transferase V (200 mU attached to beads) was added to a FBS, and 600 pg/mL (potency) of G418 (Geneticin, Sigma). The solution containing sodium cacodylate (pH 6.5, 1 M, 0.3 mL), water selection media was changed every 3 days until a stable G418-resistant (1.8 mL), MnC12 (1 M, 0.1 mL), alkaline phosphatase (32 U), GDPpopulation was established. Single clones of G418-resistant cells were fucose disodium salt (68 mg, 110 pmol), and 36 (16 mg, 9.6 pmol). isolated by cloning cylinders. Isolated clones were screened for the The reaction mixture was tipped for 21 d. Filtration and chromatogsynthesis of sol-E-selectin by enzyme-linked immunosorbent assay raphy (Biogel P-2,0.1 M W H C 0 3 ) of the filtrate afford 12 mg (68%) (ELISA) utilizing the anti-E-selectin monoclonal antibody CY 1787 as of a mixture of mono- and difucosylated products as white solid after the primary antibody. Positive clones were plated at lo6 cells/100 mm lyophilization: Rj = 0.54 (silica, 40% 1 M WOAc/2-propanol); 'H dish, 24 hours later they were metabolically labelled with [35S]NMR (D20) 6 5.13-5.10 (m, lH), 5.05-5.02 (d, J = 3.5 Hz, lH), methionene for 5 h. Labelled sol-E-selectin was immunoprecipitated 4.95-4.87 (m, 2H), 4.58-4.54 (m, 2H), 4.51-4.41 (m, 3H), 4.29from the media with CY1787 and electrophoresed through a 10% PAGE 4.25 (m, 2H), 4.24-4.20 (m, 2H), 4.07-3.44 (m, 48H), 3.30-3.22 gel, the gel dried and subjected to authoradiography. Clone 293#3 (m, lH), 2.71-2.66 (m, 2H), 2.32 (t, J = 7.4 Hz, 2H), 2.09-1.91 (m, was selected as the stable cell line that produced the greatest amount 12H), 1.76-1.68 (m, 2H), 1.59-1.49 (m, 4H), 1.33-1.25 (m, 2H), of the 110-Kd sol-E-selectin proteinkell. 1.09 (d, J = 6.5 Hz, 6H); MS (ion spray) calcd for C75H122N4052 (19) 1912, found 1912 (M+), 1766. Large Scale Production of Sol-E-selectin. A 10-chambered Nunc Cell Factory (6250 cm2total surface area, Nunc) was seeded with 2.78 5-(Methoxycarbonyl)pentyl 2,3-Bis-O-[(ammonium 5-acetamidex lo8 cells (Clone 293#3) in 850 mL in DMEM supplemented with 3J-dideoxy-a-~-g~cero-~-gu -2-nonulopyranuronat)-(2-3)-fl~c~o 5% FBS and incubated at 37 "C for 72 h. The media was harvested D-gdactopyranosyl-(l-4)-(a-L-fucopyranosyl)-(1-3)-2-acetamidoand replaced with 850 mL of DMEM, 5% FBS. After the cell factory 2-deoxy-fl-D-gdactopyranosy~]-fl-~-gdactopyranoside (18). The was incubated at 37 "C for 48 h the media was harvested a second fucosyl transferase V (200 m u attached to beads) was added to a time and replaced with 850 mL of DMEM 5 % FBS. After the cell solution containing sodium cacodylate (pH 6.5, 1 M, 0.3 mL), water factory was incubated at 37 "C for 48 h the media was harvested a (1.8 mL), MnC12 (1 M, 0.1 mL), alkaline phosphatase (32 U), GDPthird (and final) time. After each harvest 0.02% sodium azide was fucose disodium salt (68 mg, 110 pmol), and 37 (15 mg, 9.1 pmol). added to the media. The media was clarified by centrifugation (5000g), The reaction mixture was tipped for 15 d. Filtration and chromatogpassed through a 0.2 pm fiiter, and stored at 4 "C until further raphy (Biogel P-2.0.1 M N W C 0 3 ) of the filtrate afford 12 mg (74%) purification. of 18 as white solid after lyophilization: Rj = 0.54 (silica, 40% 1M NH40Acl2-propanol); 'H NMR (D2O) 6 5.05-5.00 (m, 2H), 4.58Immunoaffity Column. Monoclonal antibody CY 1787, anti 4.52 (m, lH), 4.50-4.44 (m, 2H), 4.10-3.40 (m, 57H), 2.72-2.66 E-selectin, was conjugated to protein-A Sepharose.21 Briefly, 28 mg (m, 2H), 2.34 (t, 2H, J = 6.5 Hz), 2.05-1.97 (m, 6H), 1.95 (s, 6H), of CY1787 (5 mg/mL) in PBS was mixed with 5 mL of protein-A 1.76-1.68 (m, 2H), 1.60-1.52 (m, 4H), 1.36-1.28 (m, 2H), 1.12Sepharose for 30 min at room temperature. The beads were then washed four times by centrifugation with 25 mL of 0.1 M borate buffer, 1.08 (m, 6H); MS (ion spray), calcd for C75H122N4052 1912, found 1912 pH 8.2, followed by two washes with 10 mL of 0.2 M triethanolamine, (M+), 1766. pH 8.2. The resin was then suspended in 40 mL of 0.2 M triethanoProduction of Recombinant Soluble E-Selectin Cells and Plasmid lamine buffer, pH 8.2, containing 0.02 M dimethyl pimelimidate. After DNA. The procedure for the preparation of recombinant E-selectin is reacting for 45 min at room temperature on a rotator, the resin was essentially the same as described previou~ly.'~The adenovirus washed twice with 0.02 M ethanolamine, pH 8.2, followed with three transformed human kidney cell line 293 was obtained from the ATCC washes with 10 mL of 0.1 M borate buffer, pH 8.2. Unbound antibody (CRL-1573). The 293 cells were grown as adherent cultures in DMEM, was removed by elution with 0.1 M sodium acetate buffer, pH 4.5. obtained from Whittaker Bioproducts (Walkersville, MD), supplemented Approximately 89% of the antibody applied was conjugated to the with 10% fetal calf serum, obtained from JRH Biochemical (Lenexa, protein-A Sepharose. KS). The plasmid pCDNAI, a derivative of pCDM8,l8 was obtained from Invitrogen (San Diego, CA). The plasmid pBluescript Il was Isolation of Recombinant Soluble E-Selectin. Tissue culture obtained from Stratagene (San Diego, CA). The plasmid pSV2-Neolg supernatant (2.55 L) was passed through a 0.7 cm x 1.5 cm precolumn contains the E. coli gene encoding the aminoglycoside 3'-phosphoof protein-A Sepharose connected in series to a 1.5 cm x 3 cm affiity transferase gene. When pSV2-neo is introduced into mammalian cells, column of CY1787-protein-A-Sepharoseat a flow rate of 20 mL/h. The columns were then disconnected, and the CY 1787 affinity column the transferred cells exhibit resistance to the antibiotic (3418. was washed with 20 mM Tris buffer, pH 7.5, containing 150 mM NaCl Recombinant DNA. A soluble form of E-selectin was prepared as and 2 mM CaClz until the A280 of the eluate approached zero. Bound described in the following. A 1.67 kbp DNA fragment encoding a E-selectin was eluted with 0.1 M sodium acetate buffer, pH 3.5, truncated structural gene for E-selectin was isolated by polymerase chain containing 1 mM CaCl2 using gravity flow. Fractions (1 mL) were reaction (PCR) amplification of cDNA derived from messenger RNA collected into 300 pL of 2 M Tris, pH 10. Protein containing fractions that was isolated from IL-1 activated human endothelial cells. The were pooled and dialyzed against DPBS. Following concentration on 5'-amplimer inserted a unique CL4 I restriction site 28 nucleotides an Amicon Centriprep 30 until the protein concentration was apupstream from the initiation codon of the E-selectin structural gene. proximately 1 mg/mL, the purified E-selectin was aliquoted and stored The 3'-amplimer inserted the termination codon TGA after amino acid at -80 "C. Purity was greater than 90% by SDS-PAGE. A total of residue 527 of the mature E-selectin, followed by a unique XhoI 10 mg of E-selectin was purified from 2.5 L of cell culture media. restriction site. The carboxy terminus of soluble E-selectin is located Inhibition of HL-60 Cell Binding to Sol-E-selectin. Ninety-six at the carboxy terminus of the sixth censensus repeat element, thereby deleting the transmembrane domain. The 1.67 Kbp PCR fragment was well Immunolon 2 plates (Dynatech Laboratories, Inc., Chantilly, VA, (17) Ramphal, J. Y.; Zheng, Z.-L.; Perez, C.; Walker, L. E.; DeFrees, S. A.; Gaeta, F. C. A. J. Med. Chem., in press. (18) Seed, B. Nature 1987,329, 840. (19) Southern, P.; Berg, P. J . Mol. Appl. Gen. 1983,I , 327.
(20) Kriegler, M. Gene Transfer and Expression: A Laboratory Manual; W. H. Freeman: New York, 1991. (21)Schneider, C.; Newman, R. A,; Sutherland, D. R.; Asser, U.; Greaves, M. F. J . B i d . Chem. 1982,257, 10766.
J. Am. Chem. Soc., Vol. 117. No. 1. 1995 79
Ligand Recognition by E-Selectin
cells tor peroxidase assay __+
0
EUYl
Figure 1. Schematic representation of the E-selectin inhibition analysis based on ELISA assay. For details see the Experimental Section catalog no. 011-0103655) were coated at rwm temper" with 50 pL of a 3 p#mL solution (150 mdwell) of sol-E-selectin (recombinant soluble E-selectin) in DPBS (Dulkca's phosphate buffered saline). The sol-E-selectin molecule was bound after 3 h at m m temperature. the excess coating solution was removed by three washes with DPBS containing 1% bovine serum albumin (DPBS/I% BSA). and protein binding sites on the plate were blocked with 2W pUwell of DPBS/ I % BSA for I h. Serial dilutions of inhibitors were prepared at an initial concentrationof 10 mM in DPBS and diluted in Hanks balanced salt solution containing 20 mM HEPES, pH 1.21.4, 0.2% o-glucose and I% BSA. After removing the blocking buffer. 40 pL of appropriately diluted inhibitor was added per well followed by 2 x IO' HL-60 cells (ATCC. CCL 240) in 20 pL. After 15 min at m m temperature. the plate was washed three times with Hank's balanced salt solution containing 20 mM HEPES. pH 7.2-1.4.0.2% E-glucose. I% bovine serum albumin, and I mM calcium chloride using a Molecular Devices Microplate washer (Model no. 4845-02) adjusted for slow liquid delivery. Following addition of 50 p L of cell lysis buffer (24 mM citric acid, 51 mM sodium phosphate dibasic. 0.1% nonidet P-40). the plates were incubated for 5 min on a plate shaker at r w m temperaNre. Myelopemxidasereleased frombound HL-M) cells was detected by the addition of 50 pL of substrate solution (24 mM citric acid. 5 I mM dibasic sodium phosphate. 0.1% 0-phenylenediamine and 0.03% hydrogen peroxide). The reaction was stopped with 10 min by the addition to each well of 4OpL of 4 N sulfuric acid. Absorbances
at 492 nm were determined in a TiterTex plate reader. The percentage inhibition was determined based on the absorbance in positive control wells containing no inhibitor. For a schematic representation of the assay, see Figure 7. N M R Experiments. Proton and c a h n NMR experiments were conducted in D20 at 295 K using a Bruker AMX-500 NMR spectrometer equipped with an X-32 computer and an ASPECT-3000 process controller. The sample was not spun. 'H chemical shifts were referenced to internal HOD at 4.16 ppm. and "C chemical shifts referenced to external DMSO at 39.5 ppm. All NMR data were processed and analyzcd with the Felix program (Hare Research, Woodinville. WA) run on a Sun SPARC station or a Silicon Graphics Indigo* workstation, For further experimental details. see ref 4.
Acknowledgment. W e thank Dr. Y.-C.Lin for his early work on the NMR sNdy of compound 6 and Dr. Robert Bayer for his assistance with the E-selectin isolation. This work was partially supported by the NIH (GM 44154) and NSF (CHE9310081)to C.H.W. R.L.H. acknowledges the American Cancer Society for support in the form of a postdoctoral fellowship. W.K. was supported in part as a postdoctoral fellow by the Deutsche Forschungsgesellschaft. lA9416f2P
